CHROMOSOME 3p21.3 GENES ARE TUMOR SUPPRESSORS

ABSTRACT

Tumor suppressor genes play a major role in the pathogenesis of human lung cancer and other cancers. Cytogenetic and allelotyping studies of fresh tumor and tumor-derived cell lines showed that cytogenetic changes and allele loss on the short arm of chromosome 3 (3p) are most frequently involved in about 90% of small cell lung cancers and greater than 50% of non-small cell lung cancers. A group of recessive oncogenes, Fus1, 101F6, Gene 21 (NPRL2), Gene 26 (CACNA2D2), Luca 1 (HYAL1), Luca 2 (HYAL2), PL6, 123F2 (RaSSFI), SEM A3 and Beta* (BLU), as defined by homozygous deletions in lung cancers, have been located and isolated at 3p21.3.

This application claims the benefit of U.S. Provisional Application No.60/217,112, filed Jul. 10, 2000.

The U.S. Government has rights in the invention by virtue ofP50-CA70907.

BACKGROUND OF THE INVENTION

I. Field of the Invention

The invention generally relates to the fields of molecular biology andoncology.

II. Related Art

Cancer is the result in the occurrence of multiple factors. Mutationsmay occur in proto-oncogenes that cause cellular proliferation toincrease. Mutations also may occur in tumor suppressors whose normalfunction is to regulate cellular proliferation. Mutations in DNA repairenzymes impair the ability of the cell to repair damage beforeproliferating. Tumor suppressor genes are normal genes whose absence(loss or inactivation) can lead to cancer. Tumor suppressor genes encodeproteins that slow cell growth and division. Cancer arises when there isa mutation in both alleles.

Tumor suppressor genes (TSGs) play a major role in the pathogenesis ofhuman lung cancer and other cancers. Lung cancer cells harbor mutationsand deletions in multiple known dominant and recessive oncogenes^(6,7).Known TSGs such as Rb, p53, and putative TSGs have been found atchromosome regions 3p, 5q, 6p, 8p, 9p, and 11p as well as othersites^(6,8,9). Cytogenetic and allelotyping studies of fresh lung tumorsand tumor cells showed tumor-cell allele loss at multiple sites,suggesting the existence of one or more such TSGs^(6-8,10). However,cytogenetic changes and allele loss on the short arm of chromosome 3(3p) have been shown to be most frequently involved in about 90% ofsmall cell lung cancers (SCLCs) and >50% of non-small cell lung cancers(NSCLCs)^(6,8,10,11). SCLC and NSCLC are the two treatment groups oflung tumors and are made up of four histological types. Squamous cell-,adeno-, and large cell carcinomas are in the NSCLC group. Small celllung cancer is in the SCLC group. Approximately 75% of lung tumors areNSCLCs. Metastases occur later with NSCLC than with SCLC. SCLC is one ofthe most metastatic of solid tumors⁵². In addition, similar 3p changeshave been seen in several other cancers in addition to lung, such asrenal^(12,13), breast^(14,15), head and neck¹⁶, pancreatic¹⁷, kidney¹⁸,oral¹⁹, and uterine cervical cancers^(20,21). Furthermore, a group ofTSGs, as defined by homozygous deletions in lung cancers, have beenlocated and isolated at 3p21.3 in a 450-kb region^(6,10,22-24). Studiesof lung cancer preneoplasia indicate that 3p21 allele loss is theearliest genetic abnormality in lung cancer detected so far, occurringin hyperplastic lesions; this shows that one or more 3p-recessiveoncogenes function as “gatekeepers” in the molecular pathogenesis ofmany human cancers, including lung cancer, where it is likely to beinvolved in >50% of all cases^(6,10,22-26).

Recently, human chromosome band 3p21.3 has been shown to undergooverlapping homozygous deletions in several SCLC and NSCLC lines;candidates of TSGs have been located in this critical region in severalhuman cancers, further defining a TSG region^(6,10,24,27). The evidenceshows that genes in this 3p21 critical region are involved in regulationof the telomerase-mediated cellular immortality pathway in lung, renal,and breast cancer cells^(28,29). It has also been shown that 3p deletionoccurs more frequently in the lung tumor tissues of patients who smoke.In addition, elevated sensitivity to the carcinogen benzo[a]pyrene diolepoxide at 3p21.3 has been associated with an increased risk of lungcancer, suggesting that 3p21.3 is a molecular target of carcinogens inlung cancer³¹. Despite those studies, there remains a need to furtheridentify the functions of these genes and demonstrate their involvementwith cancer.

SUMMARY OF THE INVENTION

The tumor suppressor genes at 3p21.3 are now disclosed: Gene 26(CACNA2D2)³⁴⁰, PL6, Beta* (BLU), LUCA-1 (HYAL1), LUCA-2 (HYAL2), 123F2(RASSF1), Fus1, 101F6, Gene 21 (NPRL2), and SEM A3. The function of theindividual 3p genes in suppression of tumor growth and tumorprogression, induction of apoptosis, alteration of cell cycle kinetics,and repression of telomerase activity has been characterized by theliposome- and recombinant adenoviral vector-mediated transfer of 3pgenes in vitro and in vivo. This also is the initial disclosure of theBeta* gene.

Therefore, it is an objective of the present invention to providemethods of using tumor suppressors having a chromosomal location of3p21.3. It is also an objective to provide a tumor suppressor, Beta*.Further, it is an objective to provide methods of constructingrecombinant adenovirus in which these tumor suppressors may be inserted.

An embodiment of the present invention is an isolated polynucleotideencoding a polypeptide comprising an amino acid sequence of SEQ ID NO:2.There is also provided a nucleic acid with the sequence of SEQ ID NO:1.Further provided is an isolated polypeptide comprising the amino acidsequence of SEQ ID NO:2. Another embodiment is a nucleic acid of 15 toabout 100 base pairs comprising from 15 contiguous base pairs of SEQ IDNO:1, or the complement thereof. A further embodiment includes fromabout 20, 25, 30, 40, 50 or 100 contiguous base pairs of SEQ ID NO:1, orthe complement thereof.

Another embodiment of the invention is an isolated peptide havingbetween 10 and about 50 consecutive residues of SEQ ID NO:2. Further,the peptide may comprise 15, 20, 25, or 30 consecutive residues of SEQID NO:2. In this application, “about” is defined as within + or −2 aminoacids.

Yet another embodiment is an expression cassette comprising apolynucleotide encoding a polypeptide having the sequence of SEQ IDNO:2, wherein said polynucleotide is under the control of a promoteroperable in eukaryotic cells. In another embodiment, the promoter ofthis expression cassette is heterologous to the coding sequence. Thepromoter may be a tissue specific and inducible promoter. In anotherembodiment, the expression cassette may be contained in a viral vector.The viral vector may be a retroviral vector, an adenoviral vector, andadeno-associated viral vector, a vaccinia viral vector, or a herpesviralvector. In a further embodiment the expression cassette may comprise apolyadenylation signal.

Another embodiment is a cell comprising an expression cassettecomprising a polynucleotide encoding a polypeptide having the sequenceof SEQ ID NO:2, wherein said polynucleotide is under the control of apromoter operable in eukaryotic cells, said promoter being heterologousto said polynucleotide.

Yet another embodiment of the invention is a monoclonal antibody thatbinds immunologically to a polypeptide comprising SEQ ID NO:2, or animmunologic fragment thereof. Also provided is a monoclonal antibodywith a detectable label. The label may be a fluorescent label, achemiluminescent label, a radiolabel and an enzyme. Another embodimentof the invention is a hybridoma cell that produces a monoclonal antibodythat binds immunologically to a polypeptide comprising SEQ ID NO:2, oran immunologic fragment thereof. A further embodiment is a polyclonalantisera, antibodies of which bind immunologically to a polypeptidecomprising SEQ ID NO:2, or an immunologic fragment thereof.

Yet another embodiment is a isolated and purified nucleic acid thathybridizes, under high stringency conditions, to a DNA segmentcomprising SEQ ID NO:1, or the complement thereof. In a furtherembodiment the nucleic acid is about 15, 17, 20 or 25 bases in length.

Another embodiment of the invention is a method for constructing arecombinant adenovirus comprising: (a) providing a shuttle vector, saidshuttle vector comprising an adenoviral inverted terminal repeat (ITR)sequence, an expression cassette comprising a promoter and a poly-Asequence, a transgene under the control of said promoter, and uniquerestriction sites at the 5′- and 3′-ends of theITR-promoter-transgene-poly-A segment; (b) cutting at said restrictionenzyme sites; (c) ligating the released segment into an adenoviralvector lacking the entire E1 and E3 regions and transforming theresulting vector a bacterial host cell; (d) obtaining vector from saidbacterial host cell and digesting the vector to release theE1/E3-deleted adenovirus genome; and (e) transfecting the adenovirusgenome into E1-expressing host cells. In a further embodiment, thetransgene is Gene 26 (CACNA2D2), PL6, Beta* (BLU), Luca-1 (HYAL1),Luca-2 (HYAL2), 123F2 (RASSF1), Fus1, 101F6, Gene 21 (NPRL2), or SEM A3.In another embodiment, the promoter may be a cytomegalovirus (CMV)promoter and said poly A sequence is bovine growth hormone (BGH) poly Asequence.

Yet another embodiment of the invention is a method for constructing arecombinant adenovirus comprising: (a) providing a shuttle vectorcomprising an adenoviral inverted terminal repeat (ITR) sequence, anexpression cassette comprising a promoter and poly-A signal sequence, atransgene under the control of said promoter, a tetracyclineresistance-off responsive element, and unique restriction sites at the5′ and 3′ ends of the IRT-promoter-transgene-poly-A segment; (b) cuttingat said restriction enzyme sites; (c) ligating the released segment intoan adenoviral vector comprising a tetracyclin resistant-offtransactivator gene and lacking the entire E1 and E3 regions, andtransforming the resulting vector a bacterial host cell; (d) obtainingvector from said bacterial host cell and digesting the vector to releasethe E1/E3-deleted adenovirus genome; and (e) transfecting the adenovirusgenome into E1-expressing host cells. In a further embodiment, thetransgene is Gene 26, PL6, Beta*, LUCA-1, LUCA-2, 123F2, Fus1, 101F6,Gene 21 or SEM A3. In another embodiment, the promoter may be acytomegalovirus (CMV) promoter and said poly A sequence is bovine growthhormone (BGH) poly A sequence.

In yet another embodiment, also provided is a shuttle vector comprisingan adenoviral inverted terminal repeat (ITR) sequence, an expressioncassette comprising a promoter and poly-A sequence, a TetR-Offresponsive element, and unique restriction sites at the 5′- and 3′-endsof the ITR-promoter-poly-A segment. In another embodiment of theinvention the promoter is a cytomegalovirus (CMV) promoter and said polyA sequence is bovine growth hormone (BGH) poly A sequence. Also providedis a multipurpose cloning site in said segment, positioned between saidpromoter and said poly-A sequence.

Yet another embodiment is an adenoviral vector comprising a tetracyclineresistant-off transactivator gene and lacking the entire E1 andE3-regions.

Another embodiment of the invention is a method of diagnosing cancer ina subject comprising the steps of: (i) obtaining a biological samplefrom said subject; and (ii) assessing the expression of a functionalGene 26 (CACNA2D2), PL6, Beta* (BLU), Luca-1 (HYAL1), Luca-2 (HYAL2),123F2 (RASSF1), Fus1, 101F6, Gene 21 (NPRL2), or SEM A3 product insample. In a further embodiment the sample is a tissue sample. Thetissue sample may be brain, lung, liver, spleen, kidney, lymph node,small intestine, blood cells, pancreas, colon, stomach, cervix, breast,endometrium, prostate, testicle, ovary, skin, head and neck, esophagus,oral tissue, bone marrow or blood tissue. In another embodiment, theassessing comprises detecting a nucleic acid encoding Gene 26(CACNA2D2), PL6, Beta* (BLU), Luca-1 (HYAL1), Luca-2 (HYAL2), 123F2(RASSF1), Fus1, 101F6, Gene 21 (NPRL2), or SEM A3. Detecting maycomprise amplification said nucleic acid, nucleic acid hybridization, orsequencing. In another embodiment, assessing comprises detecting a Gene26 (CACNA2D2), PL6, Beta* (BLU), Luca-1 (HYAL1), Luca-2 (HYAL2), 123F2(RASSF1), Fus1, 101F6, Gene 21 (NPRL2), or SEM A3 polypeptide. Thedetecting of a Gene 26 (CACNA2D2), PL6, Beta* (BLU), Luca-1 (HYAL1),Luca-2 (HYAL2), 123F2 (RASSF1), Fus1, 101F6, Gene 21 (NPRL2), Gene 26(CACNA2D2), PL6, Beta* (BLU), Luca-1 (HYAL1), Luca-2 (HYAL2), 123F2(RASSF1), Fus1, 101F6, Gene 21 (NPRL2), or SEM A3 polypeptide maycomprise ELISA or immunohistochemistry. In yet another embodiment, theassessing may comprise wild-type or mutant oligonucleotidehybridization, with said oligonucleotide configured in an array on achip or wafer. In another embodiment of the invention, the expression ofGene 26 (CACNA2D2), PL6, Beta* (BLU), Luca-1 (HYAL1), Luca-2 (HYAL2),123F2 (RASSF1), Fus1, 101F6, Gene 21 (NPRL2), or SEM A3 is compared withthe expression of Gene 26 (CACNA2D2), PL6, Beta* (BLU), Luca-1 (HYAL1),Luca-2 (HYAL2), 123F2 (RASSF1), Fus1, 101F6, Gene 21 (NPRL2), or SEM A3in normal samples. In another embodiment, the comparison involvesevaluating the level of Gene 26 (CACNA2D2), PL6, Beta* (BLU), Luca-1(HYAL1), Luca-2 (HYAL2), 123F2 (RASSF1), Fus1, 101F6, Gene 21 (NPRL2),SEM A3 expression.

Another embodiment is a non-human transgenic animal lacking one or bothfunctional alleles of Gene 26 (CACNA2D2), PL6, Beta* (BLU), Luca-1(HYAL1), Luca-2 (HYAL2), 123F2 (RASSF1), Fus1, 101F6, Gene 21 (NPRL2),SEM A3. Also provided is a non-human transgenic animal thatoverexpresses Gene 26 (CACNA2D2), PL6, Beta* (BLU), Luca-1 (HYAL1),Luca-2 (HYAL2), 123F2 (RASSF1), Fus1, 101F6, Gene 21 (NPRL2), Gene 26(CACNA2D2), PL6, Beta* (BLU), Luca-1 (HYAL1), Luca-2 (HYAL2), 123F2(RASSF1), Fus1, 101F6, Gene 21 (NPRL2), or SEM A3 as compared to asimilar non-transgenic animal. In a further embodiment is a non-humantransgenic animal, the genome of which comprises an expression cassettecomprising a Gene 26 (CACNA2D2), PL6, Beta* (BLU), Luca-1 (HYAL1),Luca-2 (HYAL2), 123F2 (RASSF1), Fus1, 101F6, Gene 21 (NPRL2), Gene 26(CACNA2D2), PL6, Beta* (BLU), Luca-1 (HYAL1), Luca-2 (HYAL2), 123F2(RASSF1), Fus1, 101F6, Gene 21 (NPRL2), or SEM A3 under the control ofan inducible promoter.

An embodiment of the invention is a method for suppressing growth of atumor cell comprising contacting said cell with an expression cassettecomprising: (a) a nucleic acid encoding Gene 26 (CACNA2D2), PL6, Beta*(BLU), Luca-1 (HYAL1), Luca-2 (HYAL2), 123F2 (RASSF1), Fus1, 101F6, Gene21 (NPRL2), Gene 26 (CACNA2D2), PL6, Beta* (BLU), Luca-1 (HYAL1), Luca-2(HYAL2), 123F2 (RASSF1), Fus1, 101F6, Gene 21 (NPRL2), or SEM A3; and(b) a promoter active in said tumor cell, under conditions permittingthe uptake of said nucleic acid by said tumor cell. In anotherembodiment, the tumor cell is derived from a brain tumor, lung tumor,liver tumor, spleen tumor, kidney tumor, lymph node tumor, smallintestine tumor, blood cell tumor, pancreatic tumor, colon tumor,stomach tumor, cervix tumor, breast tumor, endometrial tumor, prostatetumor, testicle tumor, ovarian tumor, skin tumor, head and neck tumor,esophageal tumor, oral tissue tumor, or bone marrow tumor. In a furtherembodiment, the nucleic acid is contained in a viral vector. The viralvector may be a retroviral vector, an adenoviral vector, andadeno-associated viral vector, a vaccinia viral vector, and aherpesviral vector. In yet another embodiment, the nucleic acid iscontained in a liposome.

Another embodiment of the invention is a method of altering thephenotype of a tumor cell comprising contacting said cell with anexpression cassette comprising: (a) a nucleic acid encoding Gene 26(CACNA2D2), PL6, Beta* (BLU), Luca-1 (HYAL1), Luca-2 (HYAL2), 123F2(RASSF1), Fus1, 101F6, Gene 21 (NPRL2), Gene 26 (CACNA2D2), PL6, Beta*(BLU), Luca-1 (HYAL1), Luca-2 (HYAL2), 123F2 (RASSF1), Fus1, 101F6, Gene21 (NPRL2), SEM A3; and (b) a promoter active in said tumor cell, underconditions permitting the uptake of said nucleic acid by said tumorcell. In another embodiment, the phenotype is selected from the groupconsisting of proliferation, migration, contact inhibition, soft agargrowth, cell cycling, invasiveness, tumorigenesis, and metastaticpotential. In yet another embodiment, the promoter is a cytomegalovirus(CMV) promoter.

Another embodiment is a method of inhibiting cancer in a subjectsuffering therefrom comprising administering to said subject anexpression cassette comprising: (a) a nucleic acid encoding Gene 26(CACNA2D2), PL6, Beta* (BLU), Luca-1 (HYAL1), Luca-2 (HYAL2), 123F2(RASSF1), Fus1, 101F6, Gene 21 (NPRL2), Gene 26 (CACNA2D2), PL6, Beta*(BLU), Luca-1 (HYAL1), Luca-2 (HYAL2), 123F2 (RASSF1), Fus1, 101F6, Gene21 (NPRL2), or SEM A3 polypeptide; and (b) a promoter active in tumorcells of said subject, whereby expression of said polypeptide inhibitssaid cancer. In a further embodiment, the subject is a human. In otherembodiments, the nucleic acid encodes Gene 26 (CACNA2D2), PL6, Beta*(BLU), Luca-1 (HYAL1), Luca-2 (HYAL2), 123F2 (RASSF1), Fus1, 101F6, Gene21 (NPRL2), or SEM A3Gene 26 (CACNA2D2), PL6, Beta* (BLU), Luca-1(HYAL1), Luca-2 (HYAL2), 123F2 (RASSF1), Fus1, 101F6, Gene 21 (NPRL2),or SEM A3. In another embodiment, the cancer is a selected from thegroup consisting of brain cancer, lung cancer, liver cancer, spleencancer, kidney cancer, lymph node cancer, small intestine cancer, bloodcell cancer, pancreatic cancer, colon cancer, stomach cancer, cervixcancer, breast cancer, endometrial cancer, prostate cancer, testiclecancer, ovarian cancer, skin cancer, head and neck cancer, esophagealcancer, oral tissue cancer, and bone marrow cancer. In yet anotherembodiment, the expression cassette is contained in a viral vector. Theviral vector may be a retroviral vector, an adenoviral vector, andadeno-associated viral vector, a vaccinia viral vector, and aherpesviral vector. In another embodiment, the expression cassette iscontained in a lipsome. In another embodiment, the expression cassettefurther comprises a poly-A sequence. The poly-A sequence may be a bovinegrowth hormone (BGH) poly-A sequence. In a further embodiment, theexpression cassette is administered intratumorally, in the tumorvasculature, local to the tumor, regional to the tumor, or systemically.

Also provided in the method of inhibiting cancer is the administering ofa chemotherapuetic agent to said subject. In another embodiment, thechemotherapeutic comprises cisplatin (CDDP), carboplatin, procarbazine,mechlorethamine, cyclophosphamide, camptothecin, ifosfamide, melphalan,chlorambucil, busulfan, nitrosurea, dactinomycin, daunorubicin,doxorubicin, bleomycin, plicomycin, mitomycin, etoposide (VP16),tamoxifen, raloxifene, estrogen receptor binding agents, taxol,gemcitabien, navelbine, farnesyl-protein tansferase inhibitors,transplatinum, 5-fluorouracil, vincristin, vinblastin and methotrexate.Also provided is the administering radiation to said subject. In anotherembodiment, the radiation is delivered local to a cancer site or iswhole body radiation. The radiation may comprise γ-rays, X-rays,accelerated protons, microwave radiation, UV radiation or the directeddelivery of radioisotopes to tumor cells. In yet another embodiment, a asecond anticancer gene may be administered to said subject. The secondanticancer gene may be a tumor suppressor. The second anticancer genemay be an inhibitor of apoptosis. In another embodiment, the secondanticancer gene is an oncogene antisense construct.

An embodiment of the invention is a method of treating a subject withcancer, comprising the step of administering to said subject a Gene 26(CACNA2D2), PL6, Beta* (BLU), Luca-1 (HYAL1), Luca-2 (HYAL2), 123F2(RASSF1), Fus1, 101F6, Gene 21 (NPRL2), SEM A3 polypeptide. In anotherembodiment, the cancer is a selected from the group consisting of braincancer, lung cancer, liver cancer, spleen cancer, kidney cancer, lymphnode cancer, small intestine cancer, blood cell cancer, pancreaticcancer, colon cancer, stomach cancer, cervix cancer, breast cancer,endometrial cancer, prostate cancer, testicle cancer, ovarian cancer,skin cancer, head and neck cancer, esophageal cancer, oral tissuecancer, and bone marrow cancer. In a further embodiment, the polypeptideis contained within a liposome. the liposome may be comprised ofN-(1-[2,3-Dioleoyloxy]propyl)-N,N,N-trimethylammonium (DOTAP) andcholesterol. In another embodiment, the subject is human.

Another embodiment of the invention is a method of screening a candidatesubstance for anti-tumor activity comprising the steps of: (i) providinga cell lacking a functional Gene 26 (CACNA2D2), PL6, Beta* (BLU), Luca-1(HYAL1), Luca-2 (HYAL2), 123F2 (RASSF1), Fus1, 101F6, Gene 21 (NPRL2),or SEM A3 polypeptide; (ii) contacting said cell with said candidatesubstance; and (iii) determining the effect of said candidate substanceon said cell. In another embodiment, the cell is a tumor cell. Inanother embodiment, the determining may comprises comparing one or morecharacteristics of the cell in the presence of said candidate substancewith the same one or more characteristics of a similar cell in theabsence of said candidate substance. In a further embodiment, thecharacteristic is selected from the group consisting of Gene 26(CACNA2D2), PL6, Beta* (BLU), Luca-1 (HYAL1), Luca-2 (HYAL2), 123F2(RASSF1), Fus1, 101F6, Gene 21 (NPRL2), SEM A3 expression, phosphataseactivity, proliferation, metastasis, contact inhibition, soft agargrowth, cell cycle regulation, tumor formation, tumor progression,metastasis and tissue invasion. In another embodiment, the candidatesubstance is a chemotherapeutic or radiotherapeutic agent. Also providedis a candidate substance selected from a small molecule library. Infurther embodiments, the cell is contacted in vitro or in vivo.

An embodiment of the invention is a method of screening a candidatesubstance for anti-tumor activity comprising the steps of: (i) providinga cell; (ii) contacting said cell with said candidate substance; and(iii) determining the effect of said candidate substance on expressionof a Gene 26 (CACNA2D2), PL6, Beta* (BLU), Luca-1 (HYAL1), Luca-2(HYAL2), 123F2 (RASSF1), Fus1, 101F6, Gene 21 (NPRL2), or SEM A3polypeptide.

Another embodiment is a method of producing a Beta* polypeptide in ahost cell comprising: (a) providing an expression cassette comprising anucleic acid encoding Beta* operably linked to an promoter active insaid host cell; (b) transferring said expression cassette into said hostcell; and (c) culturing said host cell under conditions permittingexpression of said Beta* polypeptide.

Yet another embodiment of the invention is a method of diagnosing cancerin a subject comprising the steps of: (i) obtaining a biological samplefrom said subject; and (ii) detecting hypermethylation of the promoterregion of Gene 26 (CACNA2D2), PL6, Beta* (BLU), Luca-1 (HYAL1), Luca-2(HYAL2), 123F2 (RASSF1), Fus1, 101F6, Gene 21 (NPRL2), or SEM A3.

Other objects, features and advantages of the present invention willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples, while indicating preferred embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

BRIEF SUMMARY OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein:

FIG. 1. Scheme of construction and production of recombinant adenovirususing pAd-RAP and pAd-RAP-Shuttle system.

FIG. 2. Scheme of construction of recombinant adenovirus usingpAd-RAP-Tet-Off and pAd-RAP-TRE-CMV-Shuttle. TetR-Off=tetracyclinresistant-off transactivator gene, TRE=TetR-Off responsive elements.

FIG. 3. Timing of genetic changes found in preneoplastic lesions of therespiratory epithelium associated with primary non-small cell lungcancers.

FIG. 4. Allelotyping of 3p region in DNAs from human lung cancer celllines and tumors. Filled ovals=loss of heterozygosity; openovals=retaining of alleles; and hatched ovals=homozygous deletions.

FIG. 5. Scheme of the location of the 3p21 tumor suppressor region inhuman chromosome 3p and the structure of recombinant adenoviral vectorsof 3p genes. The sizes of the individual 3p genes and theircorresponding amino acid residues, and the active tumor suppressor (TS)regions and known TSGs in the 3p are also indicated.

FIG. 6. Effects of overexpression of 3p genes on tumor cell growth inAd-3p-transduced lung cancer cells and normal human bronchial epithelialcells. MOIs were expressed as viral particles/cell (vp/c).

FIG. 7. Quantification of adenovirus-mediated 3p gene expression inH1299 cells by Real Time RT-PCR. The MOIs are expressed as viralparticles/cell (vp/c).

FIG. 8. Induction of apoptosis by overexpression of 3p genes inAd-3p-transduced lung cancer cells and normal HBEC. Apoptosis wasanalyzed by FACS with TUNEL reaction.

FIG. 9. Effect of overexpression of 3p genes on cell cycle kinetics inAd-3p-transduced human lung cancer cells A549 and H1299.

FIG. 10. Effect of overexpression of 3p genes on A549 tumor growth byintratumoral injection of Ad-3p vectors in nude mice.

FIG. 11. Effect of overexpression of 3p genes on A549 lung metastatictumor growth by systemic injection of protamine-Ad-3p vector complexesin nude mice.

FIG. 12. Map of the RASSF1 locus, transcripts, and protein domains, A)The exon-intron structure of the RASSF1 locus with the location of theCpG islands in the predicted promoter regions (the locations of whichare shown by double-headed arrows) of RASSF1A and RASSF1C. RASSF1Atranscription is predicted to come from the most centromeric promoterregion located within a CpG island and begins with exon 1A. RASSEiF alsocommences at this promoter but is missing exon iC. Transcription ofRASSF1C is predicted to begin in the most telomeric promoter region,which is approximately 2 kilobases from that of RASSF1A and begins withexon 1. Blocks represent exons; lines represent introns. B) Schematic ofthe RASSF1A transcript and predicted protein-sequence domains. Thelocation of the various primers (PKCDF, NF, R182, and R292) used forisoform-specific reverse transcription (RT)-polymerase chain reaction(PCR) analyses are indicated. Tick marks identify the exon boundaries.The potential arc homology 3 (5H3)-binding region, putativediacylglycerol (DAG)-binding domain, PEST sequence, Rasassociationdomain, and ataxia-telangiectasia-mutated (ATM) phosphorylation site arelabeled. C) Schematic of the RASSFIC transcript and predictedprotein-sequence domains. The locations of the various primers (NOX3,R182, and R292) used for isoform-specific RT-PCR analyses are indicated.D) Schematic of the RASSFIF transcript and predicted protein-sequencedomains.

FIG. 13. RASSF1A and RASSF1C messenger RNA levels detected byisoform-specific reverse transcription-polymerase chain reaction(RT-PCR) in a sampling of lung cancer cell lines (A), breast cancerlines (B), and resected lung tumors and normal human lung and breastepithelial cultures (C). All RT-PCR products were separated on 2%agarose gels and were identified by staining with ethidium bromide.Arrows indicate location of transcripts. A) Lung cancer lines tested inlanes: 1—11157; 2=11358; 3=11727; 4=11740; 5=11748; 6=11838; 7=111184;8=111299; 9=111304; 10=111437; 11=111450; 12=111770; 13=111792;14=111963; 15 111993; 16=112009; 17=112077; iS=112108; 19=11HCC44; and20=HCC78. B) Breast cancer lines tested in lanes: 1=11CC38; 2=11CC1187;3=HTB19; 4=HTB2O; 5=HTB22; 6=11TB23; 7=11TB24; 8=11TB25; 9=11TB26;10=11TB27; 11=HTB12I; 12=HTB129; 13 HTB13O; 14=HTBI31; 15=HTB132;16=H′I′B133; 17=11CC 1395; iS=11CC 1428; 19=11CC1569; 20=11CC1806; and21=11CC2157. C) Resected lung adenocarcinoma samples (ADC 1-5) andcultures of normal small-airway epithelial cells (SAECs), normal humanbronchial epithelial (NHBE) cultures, and normal human breast epithelial(NHBRE) cultures.

FIG. 14. Expression of RASSF1A after treatment of lung cancer cells with5-aza-2′-deoxycytidine (SAza-CdR). NCI-11157, a non-small-cell lungcarcinoma (NSCLC) cell line that expresses RASSF1C but not RASSFIA, wasgrown in the presence (+ lanes) and absence (− lanes) of 0.5 p.MSAza-CdR for 48 hours. Total RNA was isolated, complementary DNA wasprepared, and isoformspecific reverse transcription-polymerase chainreaction was performed for RASSF1A, RASSF 1C, andglyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a control.

FIG. 15. Methylation-specific polymerase chain reaction (PCR) for thedetection of methylated RASSF1A 5, CpG sequences in primary resectednon-small-cell lung carcinomas (NSCLCs) and their accompanying normallung tissue (upper panel), small-cell lung carcinoma (SCLC) cell lines(middle panel), and primary breast cancers (lower panel). Representativesamples are shown. For resected NSCLCs, U=results with primers specificfor unmethylated sequences; M=results with primers specific formethylated sequences. NL=normal lung tissue; T=tumor; P=results withperipheral blood lymphocyte DNA, which is unmethylated or in vitromethylated (IVMD); and 1120=negative controls with water blanks. ForSCLCs, each lane shows the PCR results for the methylated sequences froma different cell line. Lane 20 is negative control. For the breastcancers, each lane shows the PCR results for methylated sequences from adifferent sample. PCR products were separated on 2% agarose gels andbands were detected after staining with ethidium bromide.

FIG. 16. Kaplan-Meier survival curve for 107 patients with resectednon-small-cell lung carcinomas based on RASSF1A methylation status (32methylated and 75 not methylated), For the patients with unmethylatedRASSF1A alleles, the number of cases=75, censored=39, and events=36,with a mean overall survival of 52 months (95% confidence interval[CI]=44 to 59) and a median overall survival of 49 months (95% CI=44 to59); for the patients with methylated RASSF1A alleles, the number ofcases=32, censored=nine, and events=23, with a mean overall survival of37 months (95% CI=27 to 46) and a median overall survival of 28 months(95% CI=9 to 47). The log-rank test statistic for equality of survivaldistributions for RASSF1A methylation was 3.97, with df 1, P=0.0463. Thepatients at risk for each group were: RASSF1A unmethylated-12 months(n=63), 36 months (n=34), and 60 months (n=16); RASSF1A methylated-12months (n=24), 36 months (n=13), and 60 months (n=5).

FIG. 17. Effect of RASSF1A on the in vitro and in vivo growth of thenon-small-cell lung carcinoma (NSCLC) cell line NCI-111299. A)Anchorage-dependent and anchorage-independent colony formation aftertransfection of NCI-H1299 cells with the ˜ ioo empty vector (pcDNA3.1+)or peDNA3.1+ expression vectors containing wild-type p53 or RASSF1A. Foranalysis of anchorage-dependent growth, after 2 days in nonselectivegrowth medium, transfected NCI-111299 cells were diluted into 100-mm²dishes with selective medium. Transfected cells were plated in liquidmedium (for anchorage-dependent assays) or soft agar (foranchorage-independent assays) containing 800 p·g/mL of G418. Colonieswere stained with methylene blue in anchorage-dependent experimentsafter 14 days. Results represent the average of eight to 12 experimentsin liquid medium and three soft-agarexperiments. Standard deviations areshown or are less than 2%. Solid bars, anchorage-dependent growth (95%confidence interval [CI]=0 to 36 for wt-p53 (wild-type) and 52 to 60 forRASSFIA); open bars=anchorage-independent growth (95% CI=0 to 6 forwild-type (wt)-p53 and 0 to 39 for RASSFIA). B) Northern blot analysisof the RASSF1A expression in stable clones of NCI-H1299 cellstransfected with the pcDNA3.1+ vector or pcDNA3.1+ containing RASSF1Acomplementary DNA (cDNA). The vector control (vector) and four separateclones with various RASSF1A messenger RNA levels are shown. Several ofthese clones were used in the anchorage-independent growth assay shownin D. Ethidium bromide staining of the ribosomal RNA is shown as aloading control. The clones were also verified to express the RASSF1Aisoform by reverse transcription-polymerase chain reaction with the useof isoform-specific primers. C) Soft-agar (anchorage-independent) colonyformation in stable clones of NCI-111299 cells transfected with thepcDNA3.1+ vector or pcDNA3.1+ containing RASSF1A cDNA. The means andstandard deviations are shown. For each of the RASSFIA expressingclones, the 95% CI=0 to 4 for F1A.4, 2 to 16 for F1A.5, and 3 to 14 forFIA.19. D) NCI111299 cells were infected with the pBABEpuro retrovirusexpression vectors containing either the vector control or the RASSF1Aor RASSF1C cDNAs. Infected cells (10000 per plate) were suspended in0.33% agar, and the suspension was layered over a 0.5% agar base.Colonies greater than 0.2 mm in diameter were counted after 21 days. Thelower right panel shows a representative western blot, developed with arabbit antibody to the RASSF1-glutathione S-transferase fusion protein,to verify the expression of the RASSF1 proteins. C=positive controlgenerated by transient transfection of NCI-111299 cells with peDNA3.1+containing RASSF1A cDNA; V=infection of NCI-H1299 cells with theretroviral vector control (note runover from positive control;1A=infection of NCI-H1299 cells with the retroviral vector containingRASSF1A; and 1C=infection of NCI-H1299 cells with the retroviral vectorcontaining RASSF1C. E) Effect of RASSF1A on the in vivo growth ofNCI-111299 cells. Approximately 10⁷ viable NCI-H1299 cells expressingRASSF1A were injected into the flanks of each of five previouslyirradiated BALB/c (nulnu) nude mice. Tumor size was monitored overtime,and size is shown in cubic millimeters. The average volume of tumorsgrown in more than 20 mice that were given an injection ofvector-transfected NCI-H1299 cells is shown (H1299 parent). Mice thatwere given an injection of RASSFIA-infected NCI-H1299 cells grew nomeasurable tumors.

FIG. 18. Schematic representation of the location of the putative 3p21.3tumor suppressor region in human chromosome 3p and the structure of therecombinant adenoviral vectors of 3p21.3 genes. The sizes of theindividual 3p21.3 genes and their corresponding amino acid residuesdeduced from coding sequences of cDNAs, and the active tumor suppressor(TS) regions and known TSGs in the 3p are indicated. The recombinantadenoviral vectors of 3p21.3 genes (Ad-3ps) were constructed byinserting a mammalian expression cassette in which the 3p21.3 gene wasdriven by a CMV promoter and tailed with BGH poly A signal sequence intothe E1-deleted region of the replication incompetent adenovirus type 5(Ad5) genome. The relative locations of E1-deletion (ΔE1) andE3-deletion (ΔE3), the inverted repeated terminal (IRT) sequences in theAd5 genome are indicated.

FIG. 19. Effects of exogenous expression of 3p21.3 genes on tumor cellgrowth in Ad-3p-transduced human lung cancer cells and normal bronchialepithelial cells. Cells were transduced with adenoviral vectors of3p21.3 genes, 101F6, NPRL2, BLU, RASSF1C FUS1, HYAL2, and HYAL1, controlgenes, LacZ and p53, and empty vector, Ad-EV, at highest MOIs (vp/c),5000 for A549, 1000 for H1299, 5000 for H460, 2500 for H358, and 1000for HBE, respectively, and PBS alone was used as a mock control. Thecell viability was expressed as the percentage of viable adenoviralvector-transduced cells in relation to PBS-treated control cells (100%).The error bars represent standard deviations of the mean in at leastthree individual experiments. Treatments were given in quadruplicate foreach experiment. The significance of the difference in cell viabilitybetween vector-treated cells and the Ad-EV-, Ad-LacZ-, or PBS-treatedcontrols was analyzed by two-sided Student's T-test: P<0.05 was taken assignificant. The differences between the cell viability of the Ad-EV-and Ad-LacZ-transduced cells versus PBS-treated controls were notsignificant (P=0.25 to P=0.95 from different time points and celllines). The differences between the cell viability of the Ad-101F6,Ad-Fus1, and Ad-NPRL2-transduced cells versus the Ad-EV-,Ad-LacZ-transduced, or PBS-treated controls at same MOIs weresignificant in A549, H1299, and in H460 at both 3 days and 5 daysposttransduction. (P≦0.0001 to P≦0.005) but not significant in H358 andHBEC cell lines at both 3 and 5 days posttransduction (P≧0.10 to P≧0.95,from different time points and cell lines), respectively. The effects ofAd-BLU, Ad-HYAL2, and Ad-HYAL1 on cell viability were not significant inall cell lines (P>0.45) compare to those of Ad-EV and Ad-LacZ.

FIG. 20. Quantification of adenovirus-mediated 3p21.3 gene expression inH1299 cells by real-time RT-PCR. The real-time RT-PCR was performed andPCR profiles were generated by an ABI Prism 7700 Sequence Detectionsystem and equipped software (Perkin Elmer Applied Biosystems). Knownconcentrations of 13-Actin DNA were used as a standard. The H1299 cellswere transduced by adenoviral vectors of 3p21.3 genes, FUS1 (A), 101F6(B), NPRL2 (C), and HYAL1 (D) at a MOI of 1, 5, and 10 pfu/cell for 48hr, respectively, as indicated by arrows.

FIG. 21. Induction of apoptosis by exogenous expression of 3p21.3 genesin Ad-3p-transduced human NSCLC cells and normal HBECs. Apoptosis wereanalyzed by FACS, using TUNEL reaction with FITC-labeled dUTP. Cellswere transduced with adenoviral vectors of 3p21.3 genes at an MOIs(vp/c) of 5000 for A549 (A), 1000 for H1299 (B), 5000 for H460 (C), 2500for H358 (D), and 1000 for HBEC (E), respectively, and PBS, Ad-EV, andp53 were used as controls. Cell were harvested and analyzed forapoptosis at the indicated days posttransduction. The rate of apoptosisis expressed as the percentage of FITC-labeled cells in the total cellpopulation. The error bars represent standard deviations of the mean intwo or three repeated experiments with triplicate treatments and TUNELreactions for each experiment. The significance of the difference inapoptosis between vector-treated cells and the Ad-EV-, Ad-LacZ-, orPBS-treated controls was analyzed by two-sided Student's T-test. P<0.05was considered significant. The differences between the apoptosisinduced by the Ad-EV- and Ad-LacZ-transduced cells versus PBS-treatedcontrols were not significant (P=0.925 to P=0.675 from different timepoints and cell lines). The differences between the apoptosis induced inthe Ad-101F6, Ad-FUS1, and Ad-NPRL2-transduced cells versus the Ad-EV-,Ad-LacZ, or PBS-treated controls were significant in A549 and H460 cellsat both 3 days and 5 days posttransduction (P≦0.0001 to P≦0.005), andsignificant versus the Ad-EV- and PBS-treated cells in H1299 at 5 daysposttransduction (P≦0.02), but not significant in H358 and HBEC celllines at both 3 and 5 days posttransduction at all time points (P≧0.85to P≧0.95), respectively. Induction of apoptosis in Ad-p53-transducedH358 cells were significant at all time points compared to all othertreatments (P<0.0001). Induction of apoptosis in cells treated withAd-BLU, Ad-HYAL2, and Ad-HYALya11 was not significant compared to thosetreated with PBS, Ad-EV, or Ad-LacZ, in all cell lines at all timepoints (P>0.85).

FIG. 22. Effects of intratumoral administration of adenoviral vectors of3p21.3 genes on growth of human lung cancer A549 (A) and H1299 (B)subcutaneous tumors in nu/nu mice. When the tumor reached 5 to 10 mm indiameter at about 2 weeks after tumor inoculation, the tumor wasinjected with individual adenoviral vectors of 3p21.3 genes, 101F6,NPRL2, BLU, RASSF1CFUS1, HAYL2, and HYAL1 or control vectors Ad-EV,LacZ, and p53, at a dose of 5×10¹⁰ vp/tumor each in 200 μl of PBS forthree times within a week, respectively, and PBS alone was used as amock control. Results were reported as the mean±SD in 5-10 mice for eachtreatment group: Tumor volumes were normalized by the percentageincrease of tumor sizes after treatment relative to those at thebeginning of the treatment in each group. Mean tumor volumes±SE fromthese experiments are shown. ANOVA was performed to determinestatistical significance between each treatment group using a Statisticasoftware (StatSoft Inc.) and P≦0.05 was considered significant. Thedifferences betweof en the tumor volumes ofin the Ad-101F6, Ad-FUS1,Ad-NPRL2-treated mice versus in the Ad-EV- and Ad-LacZ-treated mousecontrols were statistically significant in both A549 and H1299 tumormodels (P<0.0001), and the difference in the Ad-HYAL2-treated mice wassignificant in A549 (P=0.024) but not in H1299 tumor models, after 5days from the last injection (P<0.0001), but not significant inAd-HYAL1, Ad-HYAL2, Ad-RASSF1C, and Ad-BLU-treated (P>0.05 in both A549and H1299 tumor models).

FIG. 23. Effect of systemic administration of protamine-Ad-3p complexeson development of A549 experimental lung metastases in nu/nu mice. A.,Relative metastatic tumors in mice treated with P-Ad-3p21.3 genes. Allanimals were i.v. injected with various protamine-adenoviral vectorcomplexes every other two days for 3 times each at a dose of 3×10¹⁰viral particles plus 300 μg protamine in a total volume of 200 μl peranimal, and PBS alone was used as a mock control. Each treatment groupconsisted of 5-10 animals. Lungs were harvested two weeks after the lastinjection and metastastic colonies on the surfaces of lung were countedwithout knowledge of the treatment groups. Development of metastaseswere represented as the percentages of metastatic colonies formed inprotamine-adenovirus complexes-treated groups in relation to those inthe PBS-treated group (as 100%). Error bars represent as standard error(SE). Non-parametric t-test (Wald-Wolfowitz Runs Test) was performed todetermine statistical significance between each treatment group using aStatistica software (StatSoft Inc.) and P≦0.05 was consideredsignificant. A significant inhibition of development of metastases wasobserved in mice treated with P-Ad-101F6 (P=0.002), P-Ad-NPRL2(P=0.001), P-Ad-BLU (P=0.018), P-Ad-FUS1 (P=0.002), and P-Ad-HYAL2(P=0.014), respectively, compared to mice treated with PBS, P-Ad-EV, orP-Ad-LacZ, but no significant inhibition in mice treated with .P-Ad-BLU(P=0.818) or P-Ad-HYAL1 (P=0.904). B., the representative photos oflungs stained with India ink for metastases. The metastatic colonieswere shown as white spots on the surfaces of lung.

FIG. 24. (a) RT-PCR Analysis of NSCLCs cDNA HCC515 (Wild type FUS1) andH322 (smaller cDNA mutant form of FUS1). (b) Genomic structure of wildtype FUS1 and the mutant aberrant slicing form. Top line is genomic DNAfrom cosmid clone LUCA#13 (#Z84492) and the indicated nucleotidesequence numbers. Arrowheads indicated primers for SSCP analysis. Boxesrepresent cDNA with the open reading frames (black) and untranslatedregions (white) for the 110 amino acid wild type and 82 amino acidaberrant splice form of FUS1. Note the sequence for FUS1 andFUS1-aberrant is the same for the first 80 amino acids. Three sets ofprimers were designed to cover the full FUS1 open reading frame forPCR-SSCP analysis. The primers used were S1: GTTATGGTAGTGCGGACTG andASI, GGTGGAACCATTGCCCTTAC; S2. GACCTGTGACATTTGCCGTG and AS2,CAACAGATCCCATCTGGGTC: S3; and CCTGAGCTGACCCCTTACA and AS3,TCTGTCTGCCACCTCCCAG.

FIG. 25. (a.) Western blot analysis of endogenous and transientexpression of FUS1 in lung cancer cells. Transfection was performedaccording to the manufacture's instruction using DMRIE C (LifeTechnologies, Inc., GIBCO BRL Gaithersburg, Md.). NSCLC H1299 (2×10⁵cells) were plated in 3.5 cm dishes 24 hour before transfection and 2 μgof plasmid and 4 μl of DMRIE C were used for each transfection. All ofthe plasmids were resequenced after PCR construction and the sequencesof the various FUS1 open reading frames were verified. Ten μl of lysatewas made from 2×10⁴ cells using sample buffer (100 mM Tris 2% SDS10%β-mercaptoethanol 20% glycerol 0.03% PBP) and run in 12.5% SDS-PAGE gelsfollowed by transfer to nitrocellulose membranes. After blocking with 5%dry milk and 0.2% Tween 20 in PBS, the membranes were incubated at roomtemperature for 1 h with rabbit polyclonal antibodies. Anti FUS1antibodies (1:300 dilution of sera) were generated by immunizing rabbits(Strategic Biosolution Ramona, Calif.) with peptides corresponding toamino acid 1 to 15 of the human FUS1 protein sequence. Anti-FLAGantibody M2 was from Sigma (St. Louis, Mo.). The membranes weredeveloped after incubation with presence of peroxidase-labeledanti-rabbit or anti-mouse IgG antibodies using Super Signalchemiluminescent substrate (Pierce Rockford, Ill.). The calculatedmolecular weight of FLAG-tagged FUS1 is 15 kd and the size of the bandthat was recognized by both antibodies is slightly higher than thecalculated size. As expected the mutant FUS1 (predicted to be 82 aminoacids) is slightly smaller than wild type FUS1 (110 amino acids). (b.)Results of colony formation assays in H1299 NSCLC cells. Aftertransfection, the H1299 cells were trypsinized, replated and cultured inG418 (600 μg/ml) supplemented medium (RPMI 1640 5% fetal bovine serum)for 2 or 3 weeks and the number of G418 resistant colonies counted afterstaining with methylene blue in ethanol/PBS (50/50%). Note dramaticsuppression of colony formation after transfection with FUS1 andFUS1-FLAG but much less suppression with the 82 amino acid aberrant FUS1construct. The mean and standard deviations for an average of 2-4 platesfor 2 or more experiments for H1299 were: vector control pcDNA3.1,100±18% (100%=248 colonies), FUS1-FLAG 16±10%, FUS1 23±11%, FUS1 mutant77±11%. Colony numbers of FUS1 and FUS1-FLAG transfected cells weresignificantly reduced (P<0.01, student's t test) compared with vectorcontrol. H322 cells had 40±34% colony formation with FUS1-FLAGtransfection compared to 100% for vector control (P<0.05).

FIG. 26. (a.) Induction of FUS1 protein by Ecdysone expression vector(Invitrogen, Carlsbad, Calif.) under the control of the Ponasterone A inNCI-H1299 stable transfected clones. The inventors transfected theregulatable hormone receptor vector pVgRXR into H1299 and obtained 20Zeocin (selection marker of pVgRXR) resistant clones. These stablepVgRXR transfectants were screened for β-gal activity followingtransfection with pIND-LacZ. From these clones the inventors selectedclone ECR 9 as a parent cell line in which β-gal activity wasspecifically regulated by Ponasterone A in H1299 cells. The inventorsmade an expression vector which contained FUS1-FLAG (pIND sp1-FUS1-FLAG)and transfected this into ECR 9. Western analysis. Ten μg total celllysate protein from each cell line and anti-FUS1 antibody were used forthe analysis. The concentration (μM) of Ponasterone A used for inductionis indicated above the blots. ECR9 is H1299 parent cell line transfectedwith the regulatory vector alone; clones 13 and 16 represent H1299clones containing a regulatable FUS1 vector. The in vitro growth of (b.)NSCLC H1299ECR 9 (control), (c.) H1299FUS1Clone13 and (d.)H1299FUS1Clone16 was measured by the MTT assay. Cells (10⁴) were platedin 1 ml of RPMI 1640 (Life Technologies Inc.) with 5% fetal bovine serumand cultured in the presence (1, 5 μM) or absence of Ponasterone A in a24 well plates (added at day 0) and wells were harvested for MTT assaysat the days indicated. MTT (Sigma) was added to the cultures (500μg/ml), incubated at 37° C. for 2 hours, the intracellular formazancrystals solubilized with isopropanol containing 0.01 N HCl, and theabsorbance of the solution at 560 nm was measured using aspectrophotometer. The OD 560 is directly proportioned to cell number inthe range of 0-1.2. Data points represent an average of 3 wells with SD(contained within the symbols) of each data point ˜5%. For cell cycledistribution analysis of the FUS1 inducible H1299 clones, cells (2×10⁵)of ERC 9, CL.13 and Cl. 16 were plated on 10 cm dishes and cultured inthe presence (5 μM) or absence of Ponasterone A for 2 days. Cells wereharvested, fixed in 50% ethanol/PBS, treated with 5 mg/ml RNase, stainedwith propidium iodide and analyzed for DNA content by FACSCaliberinstrument (Becton Dickinson San Jose, Calif.). FACS analysis wasperformed in three independent experiments with similar results. UnderFUS1 induced conditions the % of cells in G1 increases significantly(P<0.05) compared to controls.

SEQUENCE SUMMARY

SEQ ID NO: 1=Beta* (BLU) nucleotide sequenceSEQ ID NO: 2=Beta* (BLU) amino acid sequence

DETAILED DESCRIPTION OF THE INVENTION

Tumor suppressor genes (TSGs) play a major role in the pathogenesis ofhuman lung cancer and other cancers. Lung cancer cells harbor mutationsand deletions in multiple known dominant and recessive oncogenes^(6,7).Other TSGs that have been found to be altered in lung cancer are p53,p16, Rb, and FHIT-1⁵². Known TSGs such as Rb, p53, and others have beenfound at chromosome regions 3p, 5q, 6p, 8p, 9p, and 11p as well as othersites^(6,8,9). Cytogenetic and allelotyping studies of fresh lung tumorsand tumor cells showed tumor-cell allele loss at multiple sites,suggesting the existence of one or more such TSGs^(6-8,10). These lociare important in understanding predisposition to lung cancer amongsmokers⁵². Loss of heterozygosity (LOH) is common in lung cancers, as inother solid tumors. Some of the chromosomal loci that experience a lossof heterozygosity in lung cancer are: 9p21-p22, 13q14, 17p13.1,3p12-p14, 3p21, 3p25, 5q21, 11q12-q24, and 22q. Vulnerability to lungcancer may be due to genetic differences occurring at multiple loci.These genes may play a role in the metabolization of tobaccocarcinogens. Cytogenetic changes and allele loss on the short arm ofchromosome 3 (3p) have been shown to be most frequently involved inabout 90% of small cell lung cancers (SCLCs) and >50% of non-small celllung cancers (NSCLCs)^(6,8,10,11). In addition, similar 3p changes havebeen seen in several other cancers, such as renal^(12,13),breast^(14,15), head and neck¹⁶, pancreatic¹⁷, kidney¹⁸, oral¹⁹, anduterine cervical cancers^(20,21).

Recently, human chromosome band 3p21.3 has been shown to undergooverlapping homozygous deletions in several SCLC and NSCLC lines.Candidates of TSGs have been located in this critical region in severalhuman cancers, further defining a TSG region^(6,10,24,27). The evidenceshows that genes in this 3p21 critical region are involved in regulationof the telomerase-mediated cellular immortality pathway in lung, renal,and breast cancer cells^(28,29). Cell hybrid and microcell chromosome 3transfer studies have demonstrated the ability of human chromosome 3genes to suppress malignancy in human lung, renal, and ovarian cancercell lines^(6,30). It also has been shown that 3p deletion occurs morefrequently in the lung tumor tissues of patients who smoke. In addition,elevated sensitivity to the carcinogen benzo[a]pyrene diol epoxide at3p21.3 has been associated with an increased risk of lung cancer,suggesting that 3p21.3 can be a molecular target of carcinogens in lungcancer³¹.

This invention identifies genetic loci involved in lung cancer. A groupof TSGs (Fus1, 101F6, Gene21 (NPRL2), Gene26 (CACNA2D2), PL6, Luca1(HYAL1), Luca2 (HYAL2), 123F2 (RASSF1), Beta* (BLU) and SEM A3), asdefined by homozygous deletions in lung cancers, have been located andisolated at 3p21.3 in a 450-kb region^(6,10,22-24). Studies of lungcancer preneoplasia indicate that 3p21 allele loss is the earliestgenetic abnormality in lung cancer detected so far. One or more3p-recessive oncogenes function as “gatekeepers” in the molecularpathogenesis of many human cancers, including lung cancer, where it islikely to be involved in >50% of all cases^(6,10,22-26) (FIG. 3).

Since (1) the 3p genes located at 3p21.3 in a 450 kb region are definedby homozygous deletions in lung cancers; (2) the 3p21 allele loss is oneof the earliest genetic abnormalities detected in lung cancer and othertumors; (3) the loss of heterozygosity, the homozygous deletion, and theabnormality of these 3p genes are associated with the pathogenesis ofmany human cancers including lung cancer where it is likely to beinvolved in >50% of all cases; and (4) the multiple 3p genes function astumor suppressor genes or the 3p21.3 region as a tumor suppressorregion, the technologies and molecular tools developed based on thegenetic/cytogenetic status and function of these 3p genes are extremelyvaluable for the early detection, diagnosis, and monitoring ofprevention and therapeutic efforts for various human cancers.

I. Function of 3p Genes as Tumor Suppressor Gene Region

One of the criteria for defining the role of genes as tumor suppressorgenes is to demonstrate that the tumor phenotype marked by inactivationof the genes can be rescued by the replacement of the wild-type allelesof these genes. If the frequent loss of heterozygosity (LOH), homozygousdeletion, or, in some cases, abnormal transcripts and mutations of genesare the targets of carcinogens and the loss of function of genes leadsto human cancers, then replacement of the abnormal genes with thewild-type genes would result in tumor suppression similar to that shownby the Rb or p53 tumor suppressor gene including inhibition of tumorcell growth in vitro, suppression of tumorigenicity and tumor growth,and inhibition of tumor cell invasion and metastasis in vivo³²⁻³⁴

The identification of the 3p genes as tumor suppressor genes was basedon the cytogenetic and alleotyping studies of fresh tumors and tumorcell lines showing tumor cell allele loss at multiple sites andhomozygous deletion in this region. Some of these 3p genes share varieddegrees of homology in DNA and the predicted amino acid sequences tosome known genes in the presently available data bases; however, thefunction of these 3p genes or the 3p21.3 region in pathogenesis andtumorigenesis of cancers is previously unknown. Cell hybrid andmicrocell chromosome 3 transfer studies demonstrated the ability ofhuman chromosome 3 genes to suppress malignancy in human lung, renal,and ovarian cancer cell lines and mouse A9 fibrosarcoma cells, however,only one example involving introduction of a whole chromosome 3 intoA549 human lung carcinoma cells has been reported^(10,30,36-38).

In the present invention, it is the first time that the function of theindividual 3p genes in suppression of tumor growth and tumorprogression, induction of apoptosis, alteration of cell cycle kinetics,as well as repression of telomerase activity has been characterized bythe liposome- and the recombinant adenoviral vector-mediated transfer of3p genes in vitro and in vivo, and that the concept of function of 3pgenes as a tumor suppressor region has been developed based on the tumorsuppressor activities involved in multiple 3p genes in this critical3p21.3 region. The finding of the 3p tumor suppressors permits newtherapeutics to be developed for treating related cancers.

The adenoviral vector has been shown to be the most efficient genedelivery system in vitro and in vivo^(4,5). Recombinant adenovirusvectors have been widely used for gene transfer in basic research aswell as for clinical applications¹⁻³. However, in vitro manipulation ofadenoviral DNA is very difficult due to the large size of the genome andlimited unique and useful restriction sites, making the construction ofrecombinant adenoviral vectors relatively time consuming and laborintensive. Two conventional methods for the construction suchrecombinant adenoviruses are well documented: an in vitro ligationmethod³⁹ and an in vivo homologous recombination method⁴⁰. The in vitroligation method consists of a first step of subcloning the transgeneinto a plasmid vector to generate a segment containing the left end ofthe viral genome and a mammalian gene expression cassette, and then therecombinant vector is produced by in vitro ligation of the segment intothe viral genome, followed by transfection of the reconstitutedrecombinant viral molecule into permissive 293 cells. Hiroyuki and Kaydisclose an in vitro ligation method⁴⁵. The other methods use twoplasmids with overlaping fragments to generate the recombinant virus byhomologous recombination in 293 cells. The major limitations for thesemethods are the generation of a background of nonrecombinant virus, lowfrequency of in vivo homologous recombination, and repeated screening ofplaque to isolate pure recombinant vectors. There are severalalternative procedures for construction of recombinant adenoviralvectors based on homologous recombination of the two plasmidscotransfected in 293 cells⁴⁰, the targeted modification of theadenoviral genome in an infectious yeast artificial chromosome (YAC) inyeast cells⁴¹, the cosmid adenoviral vectors in cosmid packagingbacteria⁴², and plasmids in recA⁺ bacteria strain^(43,44). These methodswhile more efficient, are more complex, require the use of an additionalyeast hosts or nonconventional bacterial strain, face the low frequencyof homologous recombination in these host and the instability of therecombinant adenoviral genome in plasmids hosted by the recA⁺ bacterialstrain.

By comparison, the present Ad-RAP system is very simple, efficient, andrapid for the construction of recombinant adenoviral vector for genetherapy. This system requires a simple in vitro ligation using regularmolecular biology reagents and commonly used bacterial strain. Theresulting recombinant adenoviral genome containing plasmids can beeasily screened and are stable. The subsquent transfection of thelinearized recombinant adenovirus DNA mediated by liposome (DOTAP) intothe permissive 293 cells is very efficient and a homogeneous populationof recombinant adenovirus can be produced rapidly.

The recombinant adenoviral vector, Ad-3ps, can be used to deliver 3pgenes in vitro and in vivo with a much higher efficiency than any otheravailable gene delivery systems and technologies. Due to the highefficiency of transduction and high level expression of transgenes invarious cell types mediated by adenoviral vectors, the Ad-3p vectors canbe used as a effective tool to study the biological function andmechanisms of these tumor suppressor genes in vitro and in vivo. TheAd-3ps can be used to limit tumorigenicity, tumor suppression, andrestriction of metastatic processes in various tumors such as lung,colon, breast, stomach, cervix, and head and neck, prostate, andpancreas by either intravenous or intratumoral injection of the Ad-3pvector or protamine-Ad-3p complexes.

In many cases, expression of some genes such as Bak, Bax, FasL arehighly toxic to the host 293 cells, making construction and productionof the recombinant adenovirus bearing such genes extremely difficult andsome times impossible by any of the above methods and procedures. Thepresent Ad-RAP-TetR-Off system can be used to successfully construct andproduce such recombinant adenoviral vectors. The expression of thetransgene in the adenoviral vector can be turned off by addition oftetracycline into the cell culture medium, and, consequently, the toxiceffect of the gene on the host cells can be avoided and the recombinantadenovirus can be produced in the 293 cells as usual. Some other systemssuch as binary adenoviral vector systems⁴⁶ have been developed tosuccessfully construct such recombinant adenovral vectors. However, theexpression of a transgene in one viral vector depends on the expressionof a trans-activator gene in another one, i.e., two adenoviral vectorsare required for transgene expression in vitro and in vivo, which, inturn, limited the application of such a system in vivo. By comparison,in the Ad-TetR-Off vector system, the trans-activator TetR-Off gene andthe TetR-Off response element (TRE) co-exist in the same adenoviralvector, and, therefore, expression of transgene can be turned on or offin one vector in the absence or presence of the tetracycline inducer.Furthermore, since the transgene is under the control of the TREregulatory promoter, the level of expression of the transgene can beefficiently regulated by administration of tetracycline in vitro and invivo. Together, these novel features of the Ad-RAP-Tet-Off system makeit a useful new tool for rapid and successful construction andproduction of a recombinant adenoviral vector carrying cytotoxic genes.

Introduction of individual wild-type 3p21.3 genes by liposome- andadenovirus-mediated transient transfection into lung cancer cell linescontaining either heterozygous or homozygous deletion of the 3p regioninhibited tumor cell growth, induced apoptosis, and altered cell cyclekinetics, suppressed tumor growth and tumor progression in nude mice.Varied levels of inhibition of cell growth, induction of apoptosis, andalteration of cell cycle kinetics were observed in Ad-Fus1, Ad101F6, andAd-Gene 21-transduced human lung cancer cells H1299, A549, and H460,which are either lacking in 3p genes or have abnormal ones. However, nosignificant inhibitory effects on cell growth were observed in Ad-Fus1,Ad-101F6, and Ad-Gene 21-transduced normal MEC and H358 cells, whichcontain wild type 3p genes. Therefore, the observed cell growthinhibition was not due to the general cytotoxicity of these genes. Theoverexpression of 3p genes in these Ad-3p tranfectants was verified by aquantitative Real-Time RT-PCR. Tumor growth was significantly suppressedby overexpression of 101F6, Fus1, and Gene 21 via intratumoral injectionof Ad-101F6, Ad-Fus1, and Ad-Gene 21 vectors in H1299 and A549xenografts in nude mice. Furthermore, the lung metastatic tumor growthwas also significantly inhibited by systematic injection ofprotamine-complexed Ad-101F6, Ad-Fus1, and Ad-Gene 21 in nude micebearing the experimental A549 metastasis. Together, these results showthat multiple 3p genes function as tumor suppressor genes or as a tumorsuppressor region in vitro and in vivo, and that these newly identifiedand characterized 3p tumor suppressor genes or this 3p tumor suppressorregion can be used for cancer gene therapy, using molecular tools suchas the liposome-3p complexes, recombinant adenoviral vectors containing3p genes, and the local or systematic gene delivery systems developed inthis invention. The identification and functional characterization ofthe wild-type 3p21.3 genes and their mutated forms in lung cancer andother cancers provides a crucial step in the development of therapy forlung cancer and other tumors.

A. Background of 3p21.3

A group of TSGs, as defined by homozygous deletions in lung cancers,have been located and isolated at 3p21.3 in a 450-kbregion^(6,10,22-24). Studies of lung cancer preneoplasia indicate that3p21 allele loss is the earliest genetic abnormality in lung cancerdetected so far, occurring in hyperplastic lesions. One or more3p-recessive oncogenes function as “gatekeepers” in the molecularpathogenesis of many human cancers, including lung cancer, where it islikely to be involved in >50% of all cases^(6,10,22-26).

Recently, human chromosome band 3p21.3 has been shown to undergooverlapping homozygous deletions in several SCLC and NSCLC lines.Candidates of TSGs have been located in this critical region in severalhuman cancers, further defining a TSG region^(6,10,24,27). Genes in the3p21 critical region are involved in regulation of thetelomerase-mediated cellular immortality pathway in lung, renal, andbreast cancer cells^(28,29). It has also been shown that 3p deletionoccurs more frequently in the lung tumor tissues of patients who smoke.In addition, elevated sensitivity to the carcinogen benzo[a]pyrene diolepoxide at 3p21.3 has been associated with an increased risk of lungcancer, suggesting that 3p21.3 can be a molecular target of carcinogensin lung cancer³¹.

B. 3p21.3 Proteins

In addition to the entire Fus1, 101F6, Gene 21, Gene 26, Beta*, Luca1,Luca2, PL6, 123F2, and SEM A3 molecules, the present invention alsorelates to fragments of the polypeptides that may or may not retain thetumor suppressing activity. The entire length of each protein isFus1=161, 101F6=222, Gene 21-203, Gene 26=1205, Beta*=440, Luca1=435,Luca2=473, PL6=351, 123F2=431, and SEM A3=749 amino acids. Fragments,including the N-terminus of the molecule may be generated by geneticengineering of translation stop sites within the coding region(discussed below). Alternatively, treatment of the Fus1, 101F6, Gene 21,Gene 26, Beta*, Luca1, Luca2, PL6, 123F2, and SEM A3 molecules withproteolytic enzymes, known as proteases, can produce a variety ofN-terminal, C-terminal and internal fragments. Examples of fragments mayinclude contiguous residues of the Beta* sequence of 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45,50, 55, 60, 65, 75, 80, 85, 90, 95, 100, or more amino acids in length.These fragments may be purified according to known methods, such asprecipitation (e.g., ammonium sulfate), HPLC, ion exchangechromatography, affinity chromatography (including immunoaffinitychromatography) or various size separations (sedimentation, gelelectrophoresis, gel filtration).

1. Purification of 3p21.3 Proteins

It may be desirable to purify Fus1, 101F6, Gene 21, Gene 26, Beta*,Luca1, Luca2, PL6, 123F2, and SEM A3 or variants thereof. Proteinpurification techniques are well known to those of skill in the art.These techniques involve, at one level, the crude fractionation of thecellular milieu to polypeptide and non-polypeptide fractions. Havingseparated the polypeptide from other proteins, the polypeptide ofinterest may be further purified using chromatographic andelectrophoretic techniques to achieve partial or complete purification(or purification to homogeneity). Analytical methods particularly suitedto the preparation of a pure peptide are ion-exchange chromatography,exclusion chromatography; sodium dodecyl sulfate/polyacrylamide gelelectrophoresis (SDS/PAGE); isoelectric focusing. A particularlyefficient method of purifying peptides is fast protein liquidchromatography (FPLC) or even HPLC.

Various methods for quantifying the degree of purification of theprotein or peptide will be known to those of skill in the art in lightof the present disclosure. These include, for example, determining thespecific activity of an active fraction, or assessing the amount ofpolypeptides within a fraction by SDS/PAGE analysis.

Various techniques suitable for use in protein purification will be wellknown to those of skill in the art. These include, for example,precipitation with ammonium sulphate, PEG, antibodies and the like or byheat denaturation, followed by centrifugation; chromatography steps suchas ion exchange, gel filtration, reverse phase, hydroxylapatite andaffinity chromatography; isoelectric focusing; gel electrophoresis; andcombinations of such and other techniques. As is generally known in theart, it is believed that the order of conducting the variouspurification steps may be changed, or that certain steps may be omitted,and still result in a suitable method for the preparation of asubstantially purified protein or peptide.

It is known that the migration of a polypeptide can vary, sometimessignificantly, with different conditions of SDS/PAGE (Capaldi et al.,1977). It will therefore be appreciated that under differingelectrophoresis conditions, the apparent molecular weights of purifiedor partially purified expression products may vary.

High Performance Liquid Chromatography (HPLC) is characterized by a veryrapid separation with extraordinary resolution of peaks. This isachieved by the use of very fine particles and high pressure to maintainan adequate flow rate. Separation can be accomplished in a matter ofminutes, or at most an hour. Moreover, only a very small volume of thesample is needed because the particles are so small and close-packedthat the void volume is a very small fraction of the bed volume. Also,the concentration of the sample can be low because the bands are sonarrow that there is very, little dilution of the sample.

Gel chromatography, or molecular sieve chromatography, is a special typeof partition chromatography that is based on molecular size. The theorybehind gel chromatography is that the column, which is prepared withtiny particles of an inert substance that contain small pores, separateslarger molecules from smaller molecules as they pass through or aroundthe pores, depending on their size. As long as the material of which theparticles are made does not adsorb the molecules, the sole factordetermining rate of flow is the size. Hence, molecules are eluted fromthe column in decreasing size, so long as the shape is relativelyconstant. Gel chromatography is unsurpassed for separating molecules ofdifferent size because separation is independent of all other factorssuch as pH, ionic strength, temperature, etc. There also is virtually noadsorption, less zone spreading and the elution volume is related in asimple matter to molecular weight.

Affinity Chromatography is a chromatographic procedure that relies onthe specific affinity between a substance to be isolated and a moleculethat it can specifically bind to. This is a receptor-ligand typeinteraction. The column material is synthesized by covalently couplingone of the binding partners to an insoluble matrix. The column materialis then able to specifically adsorb the substance from the solution.Elution occurs by changing the conditions to those in which binding willnot occur (alter pH, ionic strength, temperature, etc.).

The matrix should be a substance that itself does not adsorb moleculesto any significant extent and that has a broad range of chemical,physical and thermal stability. The ligand should be coupled in such away as to not affect its binding properties. The ligand should alsoprovide relatively tight binding. It should be possible to elute thesubstance without destroying the sample or the ligand. One of the mostcommon forms of affinity chromatography is immunoaffinitychromatography. The generation of antibodies that would be suitable foruse in accord with the present invention is discussed below.

The present invention also describes smaller Fus1, 101F6, Gene 21, Gene26, Beta*, Luca1, Luca2, PL6, 123F2, and SEM A3-related peptides for usein various embodiments of the present invention. Because of theirrelatively small size, the peptides of the invention also can besynthesized in solution or on a solid support in accordance withconventional techniques. Various automatic synthesizers are commerciallyavailable and can be used in accordance with known protocols. See, forexample, Stewart and Young, (1984); Tam et al., (1983); Merrifield,(1986); and Barany and Merrifield (1979), each incorporated herein byreference. Short peptide sequences, or libraries of overlappingpeptides, usually from about 6 up to about 35 to 50 amino acids, whichcorrespond to the selected regions described herein, can be readilysynthesized and then screened in screening assays designed to identifyreactive peptides. Alternatively, recombinant DNA technology may beemployed wherein a nucleotide sequence which encodes a peptide of theinvention is inserted into an expression vector, transformed ortransfected into an appropriate host cell and cultivated underconditions suitable for expression.

The present invention also provides for the use of Fus1, 101F6, Gene 21,Gene 26, Beta*, Luca1, Luca2, PL6, 123F2, and SEM A3 proteins orpeptides as antigens for the immunization of animals relating to theproduction of antibodies. A biospecific or multivalent composition orvaccine is produced. It is envisioned that the methods used in thepreparation of these compositions will be familiar to those of skill inthe art and should be suitable for administration to animals, i.e.,pharmaceutically acceptable.

2. Variants of Fus1, 101F6, Gene 21, Gene 26, Beta*, Luca1, Luca2, PL6,123F2, and SEM A3

Amino acid sequence variants of these polypeptides can besubstitutional, insertional or deletion variants. Deletion variants lackone or more residues of the native protein that are not essential forfunction or immunogenic activity. Another common type of deletionvariant is one lacking secretory signal sequences or signal sequencesdirecting a protein to bind to a particular part of a cell. Insertionalmutants typically involve the addition of material at a non-terminalpoint in the polypeptide. This may include the insertion of animmunoreactive epitope or simply a single residue. Terminal additionsare called fusion proteins.

Substitutional variants typically contain the exchange of one amino acidfor another at one or more sites within the protein, and may be designedto modulate one or more properties of the polypeptide, such as stabilityagainst proteolytic cleavage, without the loss of other functions orproperties. Substitutions of this kind preferably are conservative, thatis, one amino acid is replaced with one of similar shape and charge.Conservative substitutions are well known in the art and include, forexample, the changes of: alanine to serine; arginine to lysine;asparagine to glutamine or histidine; aspartate to glutamate; cysteineto serine; glutamine to asparagine; glutamate to aspartate; glycine toproline; histidine to asparagine or glutamine; isoleucine to leucine orvaline; leucine to valine or isoleucine; lysine to arginine; methionineto leucine or isoleucine; phenylalanine to tyrosine, leucine ormethionine; serine to threonine; threonine to serine; tryptophan totyrosine; tyrosine to tryptophan or phenylalanine; and valine toisoleucine or leucine.

The following is a discussion based upon changing of the amino acids ofa protein to create an equivalent, or even an improved,second-generation molecule. For example, certain amino acids may besubstituted for other amino acids in a protein structure withoutappreciable loss of interactive binding capacity with structures suchas, for example, antigen-binding regions of antibodies or binding siteson substrate molecules. Since it is the interactive capacity and natureof a protein that defines that protein's biological functional activity,certain amino acid substitutions can be made in a protein sequence, andits underlying DNA coding sequence, and nevertheless obtain a proteinwith like properties. It is thus contemplated by the inventors thatvarious changes may be made in the DNA sequences of genes withoutappreciable loss of their biological utility or activity, as discussedbelow. Table 1 shows the codons that encode particular amino acids.

Amino acid substitutions are generally based on the relative similarityof the amino acid side-chain substituents, for example, theirhydrophobicity, hydrophilicity, charge, size, and the like. Exemplarysubstitutions that take various of the foregoing characteristics intoconsideration are well known to those of skill in the art and include:arginine and lysine; glutamate and aspartate; serine and threonine;glutamine and asparagine; and valine, leucine, and isoleucine.

C. Nucleic Acids

Fus1, 101F6, Gene 21, Gene 26, Beta*, Luca1, Luca2, PL6, 123F2, and SEMA3 are found at a chromosomal position of 3p21.3 in a 450 kb criticalregion. They are found in the following order at 3p21.3: Gene 26, PL6,101F6, Gene 21, Beta*, 123F2, Fus1, Luca2, Luca1, and SEM A3. The lengthof each is Fus1=1696, 101F6=1117, Gene 21=1696, Gene 26=5482,Beta*=1746, Luca1=2565, Luca2=1783, PL6=1860, 123F2=1502, and SEMA3=2919 nucleic acids (FIG. 5).

In addition, it should be clear that the present invention is notlimited to the specific nucleic acids disclosed herein. As discussedbelow, “Fus1, 101F6, Gene 21, Gene 26, Beta*, Luca1, Luca2, PL6, 123F2,or SEM A3 genes” may contain a variety of different bases and yet stillproduce a corresponding polypeptide that is functionallyindistinguishable, and in some cases structurally, genes disclosedherein.

Nucleic acids according to the present invention may encode an entireFus1, 101F6, Gene 21, Gene 26, Beta*, Luca1, Luca2, PL6, 123F2, and SEMA3 genes, a domain of Fus1, 101F6, Gene 21, Gene 26, Beta*, Luca1,Luca2, PL6, 123F2, and SEM A3, or any other fragment of the Fus1, 101F6,Gene 21, Gene 26, Beta*, Luca1, Luca2, PL6, 123F2, and SEM A3 sequencesset forth herein. The nucleic acid may be derived from genomic DNA,i.e., cloned directly from the genome of a particular organism. In otherembodiments, however, the nucleic acid would comprise complementary DNA(cDNA).

The term “cDNA” is intended to refer to DNA prepared using messenger RNA(mRNA) as template. The advantage of using a cDNA, as opposed to genomicDNA or DNA polymerized from a genomic, non- or partially-processed RNAtemplate, is that the cDNA primarily contains coding sequences of thecorresponding protein. There may be times when the full or partialgenomic sequence is preferred, such as where the non-coding regions arerequired for optimal expression or where non-coding regions such asintrons are to be targeted in an antisense strategy.

It also is contemplated that a given Fus1, 101F6, Gene 21, Gene 26,Beta*, Luca1, Luca2, PL6, 123F2, or SEM A3 from a given species may berepresented by natural variants that have slightly different nucleicacid sequences but, nonetheless, encode the same protein (Table 1).

As used in this application, the term “polynucleotide having the nucleicacid sequence of SEQ ID NO: 1” refers to a nucleic acid molecule thathas been isolated free of total cellular nucleic acid. A functionallyequivalent codon is a codon that encodes the same amino acid, such asthe six codons for arginine or serine (Table 1), and also refers tocodons that encode biologically equivalent amino acids.

TABLE 1 Amino Acids Codons Alanine Ala A GCA GCC GCG GCU Cysteine Cys CUGC UGU Aspartic acid Asp D GAC GAU Glutamic acid Glu E GAA GAGPhenylalanine Phe F UUC UUU Glycine Gly G GGA GGC GGG GGU Histidine HisH CAC CAU Isoleucine Ile I AUA AUC AUU Lysine Lys K AAA AAG Leucine LeuL UUA UUG CUA CUC CUG CUU Methionine Met M AUG Asparagine Asn N AAC AAUProline Pro P CCA CCC CCG CCU Glutamine Gln Q CAA CAG Arginine Arg RAGA AGG CGA CGC CGG CGU Serine Ser S AGC AGU UCA UCC UCG UCU ThreonineThr T ACA ACC ACG ACU Valine Val V GUA GUC GUG GUU Tryptophan Trp W UGGTyrosine Tyr Y UAC UAUThe DNA segments of the present invention include those encodingbiologically functional equivalent Fus1, 101F6, Gene 21, Gene 26, Beta*,Luca1, Luca2, PL6, 123F2, and SEM A3 proteins and peptides, as describedabove. Such sequences may arise as a consequence of codon redundancy andamino acid functional equivalency that are known to occur naturallywithin nucleic acid sequences and the proteins thus encoded.Alternatively, functionally equivalent proteins or peptides may becreated via the application of recombinant DNA technology, in whichchanges in the protein structure may be engineered, based onconsiderations of the properties of the amino acids being exchanged.Changes designed by man may be introduced through the application ofsite-directed mutagenesis techniques or may be introduced randomly andscreened later for the desired function, as described below.

D. Hybridization

Naturally, the present invention also encompasses DNA segments that arecomplementary, or essentially complementary, to the sequences encodingFus1, 101F6, Gene 21, Gene 26, Beta*, Luca1, Luca2, PL6, 123F2 and SEMA3. Nucleic acid sequences that are “complementary” are those that arecapable of base-pairing according to the standard Watson-Crickcomplementary rules. As used herein, the term “complementary” meansnucleic acid sequences that are substantially complementary, as may beassessed by the same nucleotide comparison set forth above, or asdefined as being capable of hybridizing to the aforementioned nucleicacid segment under relatively stringent conditions such as thosedescribed herein. Such sequences may encode the entire Fus1, 101F6, Gene21, Gene 26, Beta*, Luca1, Luca2, PL6, 123F2, and SEM A3 protein orfunctional or non-functional fragments thereof.

Alternatively, the hybridizing segments may be shorter oligonucleotides.Sequences of 17 bases long should occur only once in the human genomeand, therefore, suffice to specify a unique target sequence. Althoughshorter oligomers are easier to make and increase in vivo accessibility,numerous other factors are involved in determining the specificity ofhybridization. Both binding affinity and sequence specificity of anoligonucleotide to its complementary target increases with increasinglength. It is contemplated that exemplary oligonucleotides of 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60,65, 70, 75, 80, 85, 90, 95, 100 or more base pairs will be used,although others are contemplated. Longer polynucleotides encoding 250,500, or 1000 bases and longer are contemplated as well. Sucholigonucleotides will find use, for example, as probes in Southern andNorthern blots, in situ tissue hybridization and as primers inamplification reactions.

Accordingly, the nucleotide sequences of the invention may be used fortheir ability to selectively form duplex molecules with complementarystretches of DNAs and/or RNAs or to provide primers for amplification ofDNA or RNA from samples. Depending on the application envisioned, onewould desire to employ varying conditions of hybridization to achievevarying degrees of selectivity of the probe or primers for the targetsequence.

In certain applications, for example, substitution of amino acids bysite-directed mutagenesis, it is appreciated that lower stringencyconditions are required. Under these conditions, hybridization may occureven though the sequences of probe and target strand are not perfectlycomplementary, but are mismatched at one or more positions. Conditionsmay be rendered less stringent by increasing salt concentration anddecreasing temperature. For example, a medium stringency condition couldbe provided by about 0.1 to 0.25 M NaCl at temperatures of about 37° C.to about 55° C., while a low stringency condition could be provided byabout 0.15 M to about 0.9 M salt, at temperatures ranging from about 20°C. to about 55° C. Thus, hybridization conditions can be readilymanipulated, and thus will generally be a method of choice depending onthe desired results.

In other embodiments, hybridization may be achieved under conditions of,for example, 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mMdithiothreitol, at temperatures between approximately 20° C. to about37° C. Other hybridization conditions utilized could includeapproximately 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, attemperatures ranging from approximately 40° C. to about 72° C. Formamideand SDS also may be used to alter the hybridization conditions.

E. Primers and Probes

The term primer, as defined herein, is meant to encompass any nucleicacid that is capable of priming the synthesis of a nascent nucleic acidin a template-dependent process. Typically, primers are oligonucleotidesfrom ten to twenty base pairs in length, but longer sequences can beemployed. Primers may be provided in double-stranded or single-strandedform, although the single-stranded form is preferred. Probes are defineddifferently, although they may act as primers. Probes, while perhapscapable of priming, are designed to binding to the target DNA or RNA andneed not be used in an amplification process.

In other embodiments, the probes or primers are labeled with radioactivespecies (³²P, ¹⁴C, ³⁵S, ³H, or other label), with a fluorophore(rhodamine, fluorescein) or a chemillumiscent (luciferase).

One method of using probes and primers of the present invention is inthe search for genes related to Fus1, 101F6, Gene 21, Gene 26, Beta*,Luca1, Luca2, PL6, 123F2, and SEM A3 or, more particularly, orthologs ofFus1, 101F6, Gene 21, Gene 26, Beta*, Luca1, Luca2, PL6, 123F2, and SEMA3 from other species. Normally, the target DNA will be a genomic orcDNA library, although screening may involve analysis of RNA molecules.By varying the stringency of hybridization, and the region of the probe,different degrees of homology may be discovered.

In certain embodiments, it will be advantageous to employ nucleic acidsof defined sequences of the present invention in combination with anappropriate means, such as a label, for determining hybridization. Awide variety of appropriate indicator means are known in the art,including fluorescent, radioactive, enzymatic or other ligands, such asavidin/biotin, which are capable of being detected. In otherembodiments, one may desire to employ a fluorescent label or an enzymetag such as urease, alkaline phosphatase or peroxidase, instead ofradioactive or other environmentally undesirable reagents. In the caseof enzyme tags, colorimetric indicator substrates are known that can beemployed to provide a detection means that is visibly orspectrophotometrically detectable, to identify specific hybridizationwith complementary nucleic acid containing samples.

Another way of exploiting probes and primers of the present invention isin site-directed, or site-specific mutagenesis. Site-specificmutagenesis is a technique useful in the preparation of individualpeptides, or biologically functional equivalent proteins or peptides,through specific mutagenesis of the underlying DNA. The techniquefurther provides a ready ability to prepare and test sequence variants,incorporating one or more of the foregoing considerations, byintroducing one or more nucleotide sequence changes into the DNA.Site-specific mutagenesis allows the production of mutants through theuse of specific oligonucleotide sequences which encode the DNA sequenceof the desired mutation, as well as a sufficient number of adjacentnucleotides, to provide a primer sequence of sufficient size andsequence complexity to form a stable duplex on both sides of thedeletion junction being traversed. Typically, a primer of about 17 to 25nucleotides in length is preferred, with about 5 to 10 residues on bothsides of the junction of the sequence being altered.

In general, it is envisioned that the probes or primers described hereinwill be useful as reagents in solution hybridization, as in PCR™, fordetection of expression of corresponding genes, as well as inembodiments employing a solid phase. Representative solid phasehybridization methods are disclosed in U.S. Pat. Nos. 5,843,663,5,900,481 and 5,919,626. Other methods of hybridization that may be usedin the practice of the present invention are disclosed in U.S. Pat. Nos.5,849,481, 5,849,486 and 5,851,772. The relevant portions of these andother references identified in this section of the Specification areincorporated herein by reference.

F. Template Dependent Amplification Methods

A number of template dependent processes are available to amplify themarker sequences present in a given template sample. One of the bestknown amplification methods is the polymerase chain reaction (referredto as PCR™) which is described in detail in U.S. Pat. Nos. 4,683,195,4,683,202 and 4,800,159, and in Innis et al., 1990, each of which isincorporated herein by reference in its entirety. Other methods ofamplication are ligase chain reaction (LCR), Qbeta Replicase, isothermalamplification, strand displacement amplification (SDA), PCR™-liketemplate- and enzyme-dependent synthesis using primers with a capture ordetector moiety, transcription-based amplification systems (TAS),cylical synthesis of single-stranded and double-stranded DNA, “RACE”,one-sided PCR™, and di-oligonucleotide amplification.

Briefly, in PCR™, two primer sequences are prepared that arecomplementary to regions on opposite complementary strands of the markersequence. An excess of deoxynucleoside triphosphates are added to areaction mixture along with a DNA polymerase, e.g., Taq polymerase. Ifthe marker sequence is present in a sample, the primers will bind to themarker and the polymerase will cause the primers to be extended alongthe marker sequence by adding on nucleotides. By raising and loweringthe temperature of the reaction mixture, the extended primers willdissociate from the marker to form reaction products, excess primerswill bind to the marker and to the reaction products and the process isrepeated.

A reverse transcriptase PCR™ amplification procedure may be performed inorder to quantify the amount of mRNA amplified. Methods of reversetranscribing RNA into cDNA are well known and described in Sambrook etal., 1989. Alternative methods for reverse transcription utilizethermostable, RNA-dependent DNA polymerases. These methods are describedin WO 90/07641 filed Dec. 21, 1990. Polymerase chain reactionmethodologies are well known in the art.

G. Vectors

The term “vector” is used to refer to a carrier nucleic acid moleculeinto which a nucleic acid sequence can be inserted for introduction intoa cell where it can be replicated. A nucleic acid sequence can be“exogenous,” which means that it is foreign to the cell into which thevector is being introduced or that the sequence is homologous to asequence in the cell but in a position within the host cell nucleic acidin which the sequence is ordinarily not found. Vectors include plasmids,cosmids, viruses (bacteriophage, animal viruses, and plant viruses), andartificial chromosomes (e.g., YACs). One of skill in the art would bewell equipped to construct a vector through standard recombinanttechniques, which are described in Maniatis et al., 1988 and Ausubel etal., 1994, both incorporated herein by reference.

The term “expression cassette” refers to a vector containing a nucleicacid sequence coding for at least part of a gene product capable ofbeing transcribed. In some cases, RNA molecules are then translated intoa protein, polypeptide, or peptide. In other cases, these sequences arenot translated, for example, in the production of antisense molecules orribozymes. Expression vectors can contain a variety of “controlsequences,” which refer to nucleic acid sequences necessary for thetranscription and possibly translation of an operably linked codingsequence in a particular host organism. In addition to control sequencesthat govern transcription and translation, vectors and expressionvectors may contain nucleic acid sequences that serve other functions aswell and are described infra.

H. Promoters and Enhancers

A “promoter” is a control sequence that is a region of a nucleic acidsequence at which initiation and rate of transcription are controlled.It may contain genetic elements at which regulatory proteins andmolecules may bind such as RNA polymerase and other transcriptionfactors. The phrases “operatively positioned,” “operatively linked,”“under control,” and “under transcriptional control” mean that apromoter is in a correct functional location and/or orientation inrelation to a nucleic acid sequence to control transcriptionalinitiation and/or expression of that sequence. A promoter may or may notbe used in conjunction with an “enhancer,” which refers to a cis-actingregulatory sequence involved in the transcriptional activation of anucleic acid sequence.

A promoter may be one naturally associated with a gene or sequence, asmay be obtained by isolating the 5′ non-coding sequences locatedupstream of the coding segment and/or exon. Such a promoter can bereferred to as “endogenous.” Similarly, an enhancer may be one naturallyassociated with a nucleic acid sequence, located either downstream orupstream of that sequence. Alternatively, certain advantages will begained by positioning the coding nucleic acid segment under the controlof a recombinant or heterologous promoter, which refers to a promoterthat is not normally associated with a nucleic acid sequence in itsnatural environment. A recombinant or heterologous enhancer refers alsoto an enhancer not normally associated with a nucleic acid sequence inits natural environment. Such promoters or enhancers may includepromoters or enhancers of other genes, and promoters or enhancersisolated from any other prokaryotic, viral, or eukaryotic cell, andpromoters or enhancers not “naturally occurring,” i.e., containingdifferent elements of different transcriptional regulatory regions,and/or mutations that alter expression. In addition to producing nucleicacid sequences of promoters and enhancers synthetically, sequences maybe produced using recombinant cloning and/or nucleic acid amplificationtechnology, including PCR™, in connection with the compositionsdisclosed herein (see U.S. Pat. No. 4,683,202, U.S. Pat. No. 5,928,906,each incorporated herein by reference). Such promoters may be used todrive β-galactosidase expression for use as a reporter gene.Furthermore, it is contemplated the control sequences that directtranscription and/or expression of sequences within non-nuclearorganelles such as mitochondria, chloroplasts, and the like, can beemployed as well.

Naturally, it will be important to employ a promoter and/or enhancerthat effectively directs the expression of the DNA segment in the celltype, organelle, and organism chosen for expression. Those of skill inthe art of molecular biology generally know the use of promoters,enhancers, and cell type combinations for protein expression, forexample, see Sambrook et al., (1989), incorporated herein by reference.The promoters employed may be constitutive, tissue-specific, inducible,and/or useful under the appropriate conditions to direct high levelexpression of the introduced DNA segment, such as is advantageous in thelarge-scale production of recombinant proteins and/or peptides. Thepromoter may be heterologous or endogenous.

Table 2 lists several elements/promoters that may be employed, in thecontext of the present invention, to regulate the expression of a gene.This list is not intended to be exhaustive of all the possible elementsinvolved in the promotion of expression but, merely, to be exemplarythereof. Table 3 provides examples of inducible elements, which areregions of a nucleic acid sequence that can be activated in response toa specific stimulus.

TABLE 2 Promoter and/or Enhancer Promoter/Enhancer ReferencesImmunoglobulin Banerji et al., 1983; Gilles et al., 1983; Heavy ChainGrosschedl et al., 1985; Atchinson et al., 1986, 1987; Imler et al.,1987; Weinberger et al., 1984; Kiledjian et al., 1988; Porton et al.;1990 Immunoglobulin Queen et al., 1983; Picard et al., 1984 Light ChainT-Cell Receptor Luria et al., 1987; Winoto et al., 1989; Redondo et al.;1990 HLA DQ a and/or Sullivan et al., 1987 DQ β β-Interferon Goodbournet al., 1986; Fujita et al., 1987; Goodbourn et al., 1988 Interleukin-2Greene et al, 1989 Interleukin-2 Greene et al., 1989; Lin et al., 1990Receptor MHC Class II 5 Koch et al., 1989 MHC Class II Sherman et al.,1989 HLA-DRa β-Actin Kawamoto et al., 1988; Ng et al.; 1989 MuscleCreatine Jaynes et al., 1988; Horlick et al., 1989; Kinase (MCK) Johnsonet al., 1989 Prealbumin Costa et al., 1988 (Transthyretin) Elastase IOmitz et al., 1987 Metallothionein Karin et al., 1987; Culotta et al.,1989 (MTII) Collagenase Pinkert et al., 1987; Angel et al., 1987 AlbuminPinkert et al., 1987; Tronche et al., 1989, 1990 α-Fetoprotein Godboutet al., 1988; Campere et al., 1989 t-Globin Bodine et al., 1987;Perez-Stable et al., 1990 β-Globin Trudel et al., 1987 c-fos Cohen etal., 1987 c-HA-ras Triesman, 1986; Deschamps et al., 1985 Insulin Edlundet al., 1985 Neural Cell Hirsh et al., 1990 Adhesion Molecule (NCAM)α₁-Antitrypain Latimer et al., 1990 H2B (TH2B) Histone Hwang et al.,1990 Mouse and/or Type Ripe et al., 1989 I Collagen Glucose-RegulatedChang et al., 1989 Proteins (GRP94 and GRP78) Rat Growth Hormone Larsenet al., 1986 Human Serum Edbrooke et al., 1989 Amyloid A (SAA) TroponinI (TN I) Yutzey et al., 1989 Platelet-Derived Pech et al., 1989 GrowthFactor (PDGF) Duchenne Muscular Klamut et al., 1990 Dystrophy SV40Banerji et al., 1981; Moreau et al., 1981; Sleigh et al., 1985; Firak etal., 1986; Herr et al., 1986; Imbra et al., 1986; Kadesch et al., 1986;Wang et al., 1986; Ondek et al., 1987; Kuhl et al., 1987; Schaffner etal., 1988 Polyoma Swartzendruber et al., 1975; Vasseur et al., 1980;Katinka et al., 1980, 1981; Tyndell et al., 1981; Dandolo et al., 1983;de Villiers et al., 1984; Hen et al., 1986; Satake et al., 1988;Campbell and/or Villarreal, 1988 Retroviruses Kriegler et al., 1982,1983; Levinson et al., 1982; Kriegler et al., 1983, 1984a, b, 1988;Bosze et al., 1986; Miksicek et al., 1986; Celander et al., 1987;Thiesen et al., 1988; Celander et al., 1988; Chol et al., 1988; Reismanet al., 1989 Papilloma Campo et al., 1983; Lusky et al., 1983; VirusSpandidos and/or Wilkie, 1983; Spalholz et al., 1985; Lusky et al.,1986; Cripe et al., 1987; Gloss et al., 1987; Hirochika et al., 1987;Stephens et al., 1987; Glue et al., 1988 Hepatitis B Bulla et al., 1986;Jameel et al., 1986; Shaul Virus et al., 1987; Spandau et al., 1988;Vannice et al., 1988 Human Muesing et al., 1987; Hauber et al., 1988;Immunodeficiency Jakobovits et al., 1988; Feng et al., 1988; VirusTakebe et al., 1988; Rosen et al., 1988; Berkhout et al., 1989; Laspiaet al., 1989; Sharp et al., 1989; Braddock et al., 1989 CytomegalovirusWeber et al., 1984; Boshart et al., 1985; (CMV) Foecking et al., 1986Gibbon Ape Holbrook et al., 1987; Quinn et al., 1989 Leukemia Virus

TABLE 3 Inducible Elements Element Inducer References MT II PhorbolEster Palmiter et al., 1982; (TFA) Haslinger et al., 1985; Heavy metalsSearle et al., 1985; Stuart et al., 1985; Imagawa et al., 1987, Karin etal., 1987; Angel et al., 1987b; McNeall et al., 1989 MMTV (mouse mammaryGlucocor- Huang et al., 1981; Lee tumor virus) ticoids et al., 1981;Majors et al., 1983; Chandler et al., 1983; Lee et al., 1984; Ponta etal., 1985; Sakai et al., 1988 β-Interferon poly(rI)x Tavernier et al.,1983 poly(rc) Adenovirus 5 E2 E1A Imperiale et al., 1984 CollagenasePhorbol Ester Angel et al., 1987a (TPA) Stromelysin Phorbol Ester Angelet al., 1987b (TPA) SV40 Phorbol Ester Angel et al., 1987b (TPA) MurineMX Gene Interferon, Hug et al., 1988 Newcastle Disease Virus GRP78 GeneA23187 Resendez et al., 1988 α-2-Macroglobulin IL-6 Kunz et al., 1989Vimentin Serum Rittling et al., 1989 MHC Class I Gene H-2κb InterferonBlanar et al., 1989 HSP70 E1A, SV40 Taylor et al., 1989, 1990a, Large T1990b Antigen Proliferin Phorbol Ester- Mordacq et al., 1989 TPA TumorNecrosis Factor PMA Hensel et al., 1989 Thyroid Stimulating ThyroidChatterjee et al., 1989 Hormone α Gene Hormone

The identity of tissue-specific promoters or elements, as well as assaysto characterize their activity, is well known to those of skill in theart. Examples of such regions include the human LIMK2 gene (Nomoto etal., 1999), the somatostatin receptor 2 gene (Kraus et al., 1998),murine epididymal retinoic acid-binding gene (Lareyre et al., 1999),human CD4 (Zhao-Emonet et al., 1998), mouse alpha2 (XI) collagen(Tsumaki, et al., 1998), D1A dopamine receptor gene (Lee, et al., 1997),insulin-like growth factor II (Wu et al., 1997), human plateletendothelial cell adhesion molecule-1 (Almendro et al., 1996).

I. Initiation Signals

A specific initiation signal also may be required for efficienttranslation of coding sequences. These signals include the ATGinitiation codon or adjacent sequences. Exogenous translational controlsignals, including the ATG initiation codon; may need to be provided.One of ordinary skill in the art would readily be capable of determiningthis and providing the necessary signals. It is well known that theinitiation codon must be “in-frame” with the reading frame of thedesired coding sequence to ensure translation of the entire insert. Theexogenous translational control signals and initiation codons can beeither natural or synthetic. The efficiency of expression may beenhanced by the inclusion of appropriate transcription enhancerelements.

J. Splicing Sites

Most transcribed eukaryotic RNA molecules will undergo RNA splicing toremove introns from the primary transcripts. Vectors containing genomiceukaryotic sequences may require donor and/or acceptor splicing sites toensure proper processing of the transcript for protein expression. (SeeChandler et al., 1997, herein incorporated by reference.)

K. Polyadenylation Signals

In expression, one will typically include a polyadenylation signal toeffect proper polyadenylation of the transcript. The nature of thepolyadenylation signal is not believed to be crucial to the successfulpractice of the invention, and/or any such sequence may be employed.Specific embodiments include the SV40 polyadenylation signal and/or thebovine growth hormone polyadenylation signal, convenient and/or known tofunction well in various target cells. Also contemplated as an elementof the expression cassette is a transcriptional termination site. Theseelements can serve to enhance message levels and/or to minimize readthrough from the cassette into other sequences.

L. Origins of Replication

In order to propagate a vector in a host cell, it may contain one ormore origins of replication sites (often termed “ori”), which is aspecific nucleic acid sequence at which replication is initiated.Alternatively an autonomously replicating sequence (ARS) can be employedif the host cell is yeast.

M. Selectable and Screenable Markers

In certain embodiments of the invention, the cells contain nucleic acidconstruct of the present invention, a cell may be identified in vitro orin vivo by including a marker in the expression vector. Such markerswould confer an identifiable change to the cell permitting easyidentification of cells containing the expression vector. Generally, aselectable marker is one that confers a property that allows forselection. A positive selectable marker is one in which the presence ofthe marker allows for its selection, while a negative selectable markeris one in which its presence prevents its selection. An example of apositive selectable marker is a drug resistance marker. Examples ofselectable and screenable markers are well known to one of skill in theart.

N. Host Cells

In the context of expressing a heterologous nucleic acid sequence, “hostcell” refers to a prokaryotic or eukaryotic cell, and it includes anytransformable organisms that is capable of replicating a vector and/orexpressing a heterologous gene encoded by a vector. A host cell can, andhas been, used as a recipient for vectors. A host cell may be“transfected” or “transformed,” which refers to a process by whichexogenous nucleic acid is transferred or introduced into the host cell.A transformed cell includes the primary subject cell and its progeny.

Host cells may be derived from prokaryotes or eukaryotes, depending uponwhether the desired result is replication of the vector or expression ofpart or all of the vector-encoded nucleic acid sequences. Numerous celllines and cultures are available for use as a host cell, and they can beobtained through the American Type Culture Collection (ATCC), which isan organization that serves as an archive for living cultures andgenetic materials (www.atcc.org). An appropriate host can be determinedby one of skill in the art based on the vector backbone and the desiredresult. A plasmid or cosmid, for example, can be introduced into aprokaryote host cell for replication of many vectors. Bacterial cellsused as host cells for vector replication and/or expression includeDH5α, JM109, and KCB, as well as a number of commercially availablebacterial hosts such as SURE® Competent Cells and SOLOPACK™ Gold Cells(STRATAGENE®, La Jolla). Alternatively, bacterial cells such as E. coliLE392 could be used as host cells for phage viruses.

Examples of eukaryotic host cells for replication and/or expression of avector include HeLa, NIH3T3, Jurkat, 293, Cos, CHO, Saos, and PC12. Manyhost cells from various cell types and organisms are available and wouldbe known to one of skill in the art. Similarly, a viral vector may beused in conjunction with either a eukaryotic or prokaryotic host cell,particularly one that is permissive for replication or expression of thevector.

Some vectors may employ control sequences that allow it to be replicatedand/or expressed in both prokaryotic and eukaryotic cells. One of skillin the art would further understand the conditions under which toincubate all of the above described host cells to maintain them and topermit replication of a vector. Also understood and known are techniquesand conditions that would allow large-scale production of vectors, aswell as production of the nucleic acids encoded by vectors and theircognate polypeptides, proteins, or peptides.

O. Expression Systems

Numerous expression systems exist that comprise at least a part or allof the compositions discussed above. Prokaryote- and/or eukaryote-basedsystems can be employed for use with the present invention to producenucleic acid sequences, or their cognate polypeptides, proteins andpeptides. Many such systems are commercially and widely available.

The insect cell/baculovirus system can produce a high level of proteinexpression of a heterologous nucleic acid segment, such as described inU.S. Pat. Nos. 5,871,986, 4,879,236, both herein incorporated byreference, and which can be bought, for example, under the name MAXBAC®2.0 from INVITROGEN® and BACPACK™ B ACULOVIRUS EXPRESSION SYSTEM FROMCLONTECH®.

Other examples of expression systems include STRATAGENE®'S COMPLETECONTROL™ Inducible Mammalian Expression System, which involves asynthetic ecdysone-inducible receptor, or its pET Expression System, anE. coli expression system. Another example of an inducible expressionsystem is available from INVITROGEN®, which carries the T-REX™(tetracycline-regulated expression) System, an inducible mammalianexpression system that uses the full-length CMV promoter. I NVITROGEN®also provides a yeast expression system called the Pichia methanolicaExpression System, which is designed for high-level production ofrecombinant proteins in the methylotrophic yeast Pichia methanolica. Oneof skill in the art would know how to express a vector, such as anexpression construct, to produce a nucleic acid sequence or its cognatepolypeptide, protein, or peptide.

P. Delivery of Expression Vectors

There are a number of ways in which expression vectors may introducedinto cells. In certain embodiments of the invention, the expressionconstruct comprises a virus or engineered construct derived from a viralgenome. The ability of certain viruses to enter cells viareceptor-mediated endocytosis, to integrate into host cell genome andexpress viral genes stably and efficiently have made them attractivecandidates for the transfer of foreign genes into mammalian cells(Ridgeway, 1988; Nicolas and Rubenstein, 1988; Baichwal and Sugden,1986; Temin, 1986). The first viruses used as gene vectors were DNAviruses including the papovaviruses (simian virus 40, bovine papillomavirus, and polyoma) (Ridgeway, 1988; Baichwal and Sugden, 1986) andadenoviruses (Ridgeway, 1988; Baichwal and Sugden, 1986). These have arelatively low capacity for foreign DNA sequences and have a restrictedhost spectrum. Furthermore, their oncogenic potential and cytopathiceffects in permissive cells raise safety concerns. They can accommodateonly up to 8 kb of foreign genetic material but can be readilyintroduced in a variety of cell lines and laboratory animals (Nicolasand Rubenstein, 1988; Temin, 1986).

One of the methods for in vivo delivery involves the use of anadenovirus expression vector. “Adenovirus expression vector” is meant toinclude those constructs containing adenovirus sequences sufficient to(a) support packaging of the construct and (b) to express an antisensepolynucleotide that has been cloned therein. In this context, expressiondoes not require that the gene product be synthesized.

1. Adenovirus Expression Vectors

The expression vector comprises a genetically engineered form ofadenovirus. Knowledge of the genetic organization of adenovirus, a 36kb, linear, double-stranded DNA virus, allows substitution of largepieces of adenoviral DNA with foreign sequences up to 7 kb (Grunhaus andHorwitz, 1992). In contrast to retrovirus, the adenoviral infection ofhost cells does not result in chromosomal integration because adenoviralDNA can replicate in an episomal manner without potential genotoxicity.Also, adenoviruses are structurally stable, and no genome rearrangementhas been detected after extensive amplification. Adenovirus can infectvirtually all epithelial cells regardless of their cell cycle stage.

In one system, recombinant adenovirus is generated from homologousrecombination between shuttle vector and provirus vector. Due to thepossible recombination between two proviral vectors, wild-typeadenovirus may be generated from this process. Therefore, it is criticalto isolate a single clone of virus from an individual plaque and examineits genomic structure.

Generation and propagation of the current adenovirus vectors, which arereplication deficient, depend on a unique helper cell line, designated293, which was transformed from human embryonic kidney cells by Ad5 DNAfragments and constitutively expresses E1 proteins (Graham et al.,1977).

Helper cell lines may be derived from human cells such as humanembryonic kidney cells, muscle cells, hematopoietic cells or other humanembryonic mesenchymal or epithelial cells. Alternatively, the helpercells may be derived from the cells of other mammalian species that arepermissive for human adenovirus. Such cells include, e.g., Vero cells orother monkey embryonic mesenchymal or epithelial cells. As stated above,the preferred helper cell line is 293.

Other than the requirement that the adenovirus vector be replicationdefective, or at least conditionally defective, the nature of theadenovirus vector is not believed to be crucial to the successfulpractice of the invention. The adenovirus may be of any of the 42different known serotypes or subgroups A-F. Adenovirus type 5 ofsubgroup C is the preferred starting material in order to obtain theconditional replication-defective adenovirus vector for use in thepresent invention. This is because Adenovirus type 5 is a humanadenovirus about which a great deal of biochemical and geneticinformation is known, and it has historically been used for mostconstructions employing adenovirus as a vector.

Adenovirus vectors have been used in eukaryotic gene expression (Levreroet al., 1991; Gomez-Foix et al., 1992) and vaccine development (Grunhausand Horwitz, 1992; Graham and Prevec, 1992). Recently, animal studiessuggested that recombinant adenovirus could be used for gene therapy(Stratford-Perricaudet and Perricaudet, 1991; Stratford-Perricaudet etal., 1990; Rich et al., 1993). Studies in administering recombinantadenovirus to different tissues include trachea instillation (Rosenfeldet al., 1991; Rosenfeld et al., 1992), muscle injection (Ragot et al.,1993), peripheral intravenous injections (Herz and Gerard, 1993) andstereotactic inoculation into the brain (Le Gal La Salle et al., 1993).

2. Retrovirus Expression Vectors

The retroviruses are a group of single-stranded RNA virusescharacterized by an ability to convert their RNA to double-stranded DNAin infected cells by a process of reverse-transcription (Coffin, 1990).The resulting DNA then stably integrates into cellular chromosomes as aprovirus and directs synthesis of viral proteins. The integrationresults in the retention of the viral gene sequences in the recipientcell and its descendants. The retroviral genome contains three genes,gag, pol, and env that code for capsid proteins, polymerase enzyme, andenvelope components, respectively. A sequence found upstream from thegag gene contains a signal for packaging of the genome into virions. Twolong terminal repeat (LTR) sequences are present at the 5′ and 3′ endsof the viral genome. These contain strong promoter and enhancersequences and are also required for integration in the host cell genome(Coffin, 1990).

In order to construct a retroviral vector, a nucleic acid encoding agene of interest is inserted into the viral genome in the place ofcertain viral sequences to produce a virus that isreplication-defective. In order to produce virions, a packaging cellline containing the gag, pol, and env genes but without the LTR andpackaging components is constructed (Mann et al., 1983). When arecombinant plasmid containing a cDNA, together with the retroviral LTRand packaging sequences is introduced into this cell line (by calciumphosphate precipitation for example), the packaging sequence allows theRNA transcript of the recombinant plasmid to be packaged into viralparticles, which are then secreted into the culture media (Nicolas andRubenstein, 1988; Temin, 1986; Mann et al., 1983). The media containingthe recombinant retroviruses is then collected, optionally concentrated,and used for gene transfer. Retroviral vectors are able to infect abroad variety of cell types. However, integration and stable expressionrequire the division of host cells (Paskind et al., 1975).

3. Other Viral Vectors

Other viral vectors may be employed as expression constructs in thepresent invention. Vectors derived from viruses such as vaccinia virus(Ridgeway, 1988; Baichwal and Sugden, 1986; Coupar et al., 1988)adeno-associated virus (AAV) (Ridgeway, 1988; Baichwal and Sugden, 1986;Hermonat and Muzycska, 1984) and herpesviruses may be employed. Theyoffer several attractive features for various mammalian cells(Friedmann, 1989; Ridgeway, 1988; Baichwal and Sugden, 1986; Coupar etal., 1988; Harwich et al., 1990).

In order to effect expression of sense or antisense gene constructs, theexpression construct must be delivered into a cell. This delivery may beaccomplished in vitro, as in laboratory procedures for transformingcells lines, or in vivo or ex vivo, as in the treatment of certaindisease states. One mechanism for delivery is via viral infection wherethe expression construct is encapsidated in an infectious viralparticle.

4. Non-Viral Methods for Transfer of Expression Constructs

Several non-viral methods for the transfer of expression constructs intocultured mammalian cells also are contemplated by the present invention.These include calcium phosphate precipitation (Graham and Van Der Eb,1973; Chen and Okayama, 1987; Rippe et al., 1990) DEAE-dextran (Gopal,1985), electroporation (Tur-Kaspa et al., 1986; Potter et al., 1984),direct microinjection (Harland and Weintraub, 1985), DNA-loadedliposomes (Nicolau and Sene, 1982; Fraley et al., 1979) andlipofectamine-DNA complexes, cell sonication (Fechheimer et al., 1987),gene bombardment using high velocity microprojectiles (Yang et al.,1990), and receptor-mediated transfection (Wu and Wu, 1987; Wu and Wu,1988). Some of these techniques may be successfully adapted for in vivoor ex vivo use.

Once the expression construct has been delivered into the cell thenucleic acid encoding the gene of interest may be positioned andexpressed at different sites. In certain embodiments, the nucleic acidencoding the gene may be stably integrated into the genome of the cell.This integration may be in the cognate location and orientation viahomologous recombination (gene replacement) or it may be integrated in arandom, non-specific location (gene augmentation). In yet furtherembodiments, the nucleic acid may be stably maintained in the cell as aseparate, episomal segment of DNA. Such nucleic acid segments or“episomes” encode sequences sufficient to permit maintenance andreplication independent of or in synchronization with the host cellcycle. How the expression construct is delivered to a cell and where inthe cell the nucleic acid remains is dependent on the type of expressionconstruct employed.

In yet another embodiment of the invention, the expression construct maysimply consist of naked recombinant DNA or plasmids. Transfer of theconstruct may be performed by any of the methods mentioned above whichphysically or chemically permeabilize the cell membrane. This isparticularly applicable for transfer in vitro but it may be applied toin vivo use as well. Dubensky et al. (1984) successfully injectedpolyomavirus DNA in the form of calcium phosphate precipitates intoliver and spleen of adult and newborn mice demonstrating active viralreplication and acute infection. Benvenisty and Neshif (1986) alsodemonstrated that direct intraperitoneal injection of calciumphosphate-precipitated plasmids results in expression of the transfectedgenes. It is envisioned that DNA encoding a gene of interest also may betransferred in a similar manner in vivo and express the gene product.

In still another embodiment, the transferring a naked DNA expressionconstruct into cells may involve particle bombardment. This methoddepends on the ability to accelerate DNA-coated microprojectiles to ahigh velocity allowing them to pierce cell membranes and enter cellswithout killing them (Klein et al., 1987). Several devices foraccelerating small particles have been developed. One such device relieson a high voltage discharge to generate an electrical current, which inturn provides the motive force (Yang et al., 1990). The microprojectilesused have consisted of biologically inert substances such as tungsten orgold beads.

Selected organs including the liver, skin, and muscle tissue of rats andmice have been bombarded in vivo (Yang et al., 1990; Zelenin et al.,1991). This may require surgical exposure of the tissue or cells, toeliminate any intervening tissue between the gun and the target organ,i.e., ex vivo treatment. Again, DNA encoding a particular gene may bedelivered via this method and still be incorporated by the presentinvention.

In a further embodiment of the invention, the expression construct maybe entrapped in a liposome. Liposomes are vesicular structurescharacterized by a phospholipid bilayer membrane and an inner aqueousmedium. Multilamellar liposomes have multiple lipid layers separated byaqueous medium. They form spontaneously when phospholipids are suspendedin an excess of aqueous solution. The lipid components undergoself-rearrangement before the formation of closed structures and entrapwater and dissolved solutes between the lipid bilayers (Ghosh andBachhawat, 1991). Also contemplated are lipofectamine-DNA complexes.

Liposome-mediated nucleic acid delivery and expression of foreign DNA invitro has been very successful. Wong et al., (1980) demonstrated thefeasibility of liposome-mediated delivery and expression of foreign DNAin cultured chick embryo, HeLa and hepatoma cells. Nicolau et al.,(1987) accomplished successful liposome-mediated gene transfer in ratsafter intravenous injection.

In certain embodiments of the invention, the liposome may be complexedwith a hemagglutinating virus (HVJ). This has been shown to facilitatefusion with the cell membrane and promote cell entry ofliposome-encapsulated DNA (Kaneda et al., 1989). In other embodiments,the liposome may be complexed or employed in conjunction with nuclearnon-histone chromosomal proteins (HMG-1) (Kato et al., 1991). In yetfurther embodiments, the liposome may be complexed or employed inconjunction with both HVJ and HMG-1. In that such expression constructshave been successfully employed in transfer and expression of nucleicacid in vitro and in vivo, then they are applicable for the presentinvention. Where a bacterial promoter is employed in the DNA construct,it also will be desirable to include within the liposome an appropriatebacterial polymerase.

Other expression constructs which can be employed to deliver a nucleicacid encoding a particular gene into cells are receptor-mediateddelivery vehicles. These take advantage of the selective uptake ofmacromolecules by receptor-mediated endocytosis in almost all eukaryoticcells. Because of the cell type-specific distribution of variousreceptors, the delivery can be highly specific (Wu and Wu, 1993).

Receptor-mediated gene targeting vehicles generally consist of twocomponents: a cell receptor-specific ligand and a DNA-binding agent.Several ligands have been used for receptor-mediated gene transfer. Themost extensively characterized ligands are asialoorosomucoid (ASOR) (Wuand Wu, 1987) and transferrin (Wagner et al., 1990). Recently, asynthetic neoglycoprotein, which recognizes the same receptor as ASOR,has been used as a gene delivery vehicle (Ferkol et al., 1993; Peraleset al., 1994) and epidermal growth factor (EGF) has also been used todeliver genes to squamous carcinoma cells (Myers, EPO 0273085).

In other embodiments, the delivery vehicle may comprise a ligand and aliposome. For example, Nicolau et al., (1987) employedlactosyl-ceramide, a galactose-terminal asialganglioside, incorporatedinto liposomes and observed an increase in the uptake of the insulingene by hepatocytes. Thus, it is feasible that a nucleic acid encoding aparticular gene also may be specifically delivered into a cell type suchas lung, epithelial or tumor cells, by any number of receptor-ligandsystems with or without liposomes. For example, epidermal growth factor(EGF) may be used as the receptor for mediated delivery of a nucleicacid encoding a gene in many tumor cells that exhibit upregulation ofEGF receptor. Mannose can be used to target the mannose receptor onliver cells. Also, antibodies to CD5 (CLL), CD22 (lymphoma), CD25(T-cell leukemia) and MAA (melanoma) can similarly be used as targetingmoieties.

In certain embodiments, gene transfer may more easily be performed underex vivo conditions. Ex vivo gene therapy refers to the isolation ofcells from an animal, the delivery of a nucleic acid into the cells invitro, and then the return of the modified cells back into an animal.This may involve the surgical removal of tissue/organs from an animal orthe primary culture of cells and tissues.

Primary mammalian cell cultures may be prepared in various ways. Inorder for the cells to be kept viable while in vitro and in contact withthe expression construct, it is necessary to ensure that the cellsmaintain contact with the correct ratio of oxygen and carbon dioxide andnutrients but are protected from microbial contamination. Cell culturetechniques are well documented and are disclosed herein by reference(Freshner, 1992).

One embodiment of the foregoing involves the use of gene transfer toimmortalize cells for the production of proteins. The gene for theprotein of interest may be transferred as described above intoappropriate host cells followed by culture of cells under theappropriate conditions. The gene for virtually any polypeptide may beemployed in this manner. The generation of recombinant expressionvectors, and the elements included therein, are discussed above.Alternatively, the protein to be produced may be an endogenous proteinnormally synthesized by the cell in question.

Examples of useful mammalian host cell lines are Vero and HeLa cells andcell lines of Chinese hamster ovary, W138, BHK, COS-7, 293, HepG2,NIH3T3, RIN and MDCK cells. In addition, a host cell strain may bechosen that modulates the expression of the inserted sequences, ormodifies and process the gene product in the manner desired. Suchmodifications (e.g., glycosylation) and processing (e.g., cleavage) ofprotein products may be important for the function of the protein.Different host cells have characteristic and specific mechanisms for thepost-translational processing and modification of proteins. Appropriatecell lines or host systems can be chosen to insure the correctmodification and processing of the foreign protein expressed.

A number of selection systems may be used including, but not limited to,HSV thymidine kinase, hypoxanthine-guanine phosphoribosyltransferase andadenine phosphoribosyltransferase genes, in tk-, hgprt- or aprt-cells,respectively. Also, anti-metabolite resistance can be used as the basisof selection for dhfr, that confers resistance to; gpt, that confersresistance to mycophenolic acid; neo, that confers resistance to theaminoglycoside G418; and hygro, that confers resistance to hygromycin.

Animal cells can be propagated in vitro in two modes: as non-anchoragedependent cells growing in suspension throughout the bulk of the cultureor as anchorage-dependent cells requiring attachment to a solidsubstrate for their propagation (i.e., a monolayer type of cell growth).

Q. Antibodies

The antibodies of the present invention are useful for the isolation ofantigens by immunoprecipitation. Immunoprecipitation involves theseparation of the target antigen component from a complex mixture, andis used to discriminate or isolate minute amounts of protein. For theisolation of membrane proteins cells must be solubilized into detergentmicelles. Nonionic salts are preferred, since other agents such as bilesalts, precipitate at acid pH or in the presence of bivalent cations.Antibodies are and their uses are discussed further below.

In another aspect, the present invention contemplates an antibody thatis immunoreactive with a Fus1, 101F6, Gene 21, Gene 26, Beta*, Luca1,Luca2, PL6, 123F2, or SEM A3 molecule of the present invention, or anyportion thereof. An antibody can be a polyclonal or a monoclonalantibody. In one embodiment, an antibody is a monoclonal antibody. Meansfor preparing and characterizing antibodies are well known in the art(see, e.g., Howell and Lane, 1988).

Briefly, a polyclonal antibody is prepared by immunizing an animal withan immunogen comprising a polypeptide of the present invention andcollecting antisera from that immunized animal. A wide range of animalspecies can be used for the production of antisera. Typically an animalused for production of anti-antisera is a non-human animal includingrabbits, mice, rats, hamsters, pigs or horses. Because of the relativelylarge blood volume of rabbits, a rabbit is a preferred choice forproduction of polyclonal antibodies.

Antibodies, both polyclonal and monoclonal, specific for isoforms ofantigen may be prepared using conventional immunization techniques, aswill be generally known to those of skill in the art. A compositioncontaining antigenic epitopes of the compounds of the present inventioncan be used to immunize one or more experimental animals, such as arabbit or mouse, which will then proceed to produce specific antibodiesagainst the compounds of the present invention. Polyclonal antisera maybe obtained, after allowing time for antibody generation, simply bybleeding the animal and preparing serum samples from the whole blood.

It is proposed that the monoclonal antibodies of the present inventionwill find useful application in standard immunochemical procedures, suchas ELISA and Western blot methods and in immunohistochemical proceduressuch as tissue staining, as well as in other procedures which mayutilize antibodies specific to Fus1, 101F6, Gene 21, Gene 26, Beta*,Luca1, Luca2, PL6, 123F2, or SEM A3-related antigen epitopes.Additionally, it is proposed that monoclonal antibodies specific to theparticular Fus1, 101F6, Gene 21, Gene 26, Beta*, Luca1, Luca2, PL6,123F2, or SEM A3 of different species may be utilized in other usefulapplications

In general, both polyclonal and monoclonal antibodies against Fus1,101F6, Gene 21, Gene 26, Beta*, Luca1, Luca2, PL6, 123F2, or SEM A3 maybe used in a variety of embodiments. For example, they may be employedin antibody cloning protocols to obtain cDNAs or genes encoding otherFus1, 101F6, Gene 21, Gene 26, Beta*, Luca1, Luca2, PL6, 123F2, or SEMA3. They may also be used in inhibition studies to analyze the effectsof Fus1, 101F6, Gene 21, Gene 26, Beta*, Luca1, Luca2, PL6, 123F2, orSEM A3 related peptides in cells or animals. Anti-Fus1, 101F6, Gene 21,Gene 26, Beta*, Luca1, Luca2, PL6, 123F2, or SEM A3 antibodies also willbe useful in immunolocalization studies to analyze the distribution ofFus1, 101F6, Gene 21, Gene 26, Beta*, Luca1, Luca2, PL6, 123F2, and SEMA3 during various cellular events, for example, to determine thecellular or tissue-specific distribution of Fus1, 101F6, Gene 21, Gene26, Beta*, Luca1, Luca2, PL6, 123F2, and SEM A3 polypeptides underdifferent points in the cell cycle. A particularly useful application ofsuch antibodies is in purifying native or recombinant Fus1, 101F6, Gene21, Gene 26, Beta*, Luca1, Luca2, PL6, 123F2, or SEM A3, for example,using an antibody affinity column. The operation of all suchimmunological techniques will be known to those of skill in the art inlight of the present disclosure.

Means for preparing and characterizing antibodies are well known in theart (see, e.g., Harlow and Lane, 1988; incorporated herein byreference). More specific examples of monoclonal antibody preparationare give in the examples below.

As is well known in the art, a given composition may vary in itsimmunogenicity. It is often necessary therefore to boost the host immunesystem, as may be achieved by coupling a peptide or polypeptideimmunogen to a carrier. Exemplary and preferred carriers are keyholelimpet hemocyanin (KLH) and bovine serum albumin (BSA). Other albuminssuch as ovalbumin, mouse serum albumin or rabbit serum albumin can alsobe used as carriers. Means for conjugating a polypeptide to a carrierprotein are well known in the art and include glutaraldehyde,m-maleimidobencoyl-N-hydroxysuccinimide ester, carbodiimide andbis-biazotized benzidine.

As also is well known in the art, the immunogenicity of a particularimmunogen composition can be enhanced by the use of non-specificstimulators of the immune response, known as adjuvants. Exemplary andpreferred adjuvants include complete Freund's adjuvant (a non-specificstimulator of the immune response containing killed Mycobacteriumtuberculosis), incomplete Freund's adjuvants and aluminum hydroxideadjuvant.

The amount of immunogen composition used in the production of polyclonalantibodies varies upon the nature of the immunogen as well as the animalused for immunization. A variety of routes can be used to administer theimmunogen (subcutaneous, intramuscular, intradermal, intravenous andintraperitoneal). The production of polyclonal antibodies may bemonitored by sampling blood of the immunized animal at various pointsfollowing immunization. A second, booster, injection may also be given.The process of boosting and titering is repeated until a suitable titeris achieved. When a desired level of immunogenicity is obtained, theimmunized animal can be bled and the serum isolated and stored, and/orthe animal can be used to generate mAbs.

MAbs may be readily prepared through use of well-known techniques, suchas those exemplified in U.S. Pat. No. 4,196,265, incorporated herein byreference. Typically, this technique involves immunizing a suitableanimal with a selected immunogen composition, e.g., a purified orpartially purified Fus1, 101F6, Gene 21, Gene 26, Beta*, Luca1, Luca2,PL6, 123F2, or SEM A3 protein, polypeptide or peptide or cell expressinghigh levels of Fus1, 101F6, Gene 21, Gene 26, Beta*, Luca1, Luca2, PL6,123F2, or SEM A3. The immunizing composition is administered in a mannereffective to stimulate antibody-producing cells. Rodents such as miceand rats are preferred animals, however, the use of rabbit, sheep, andfrog cells is also possible. The use of rats may provide certainadvantages (Goding, 1986), but mice are preferred, with the BALB/c mousebeing most preferred as this is most routinely used and generally givesa higher percentage of stable fusions.

Following immunization, somatic cells with the potential for producingantibodies, specifically B-lymphocytes (B-cells), are selected for usein the mAb generating protocol. These cells may be obtained frombiopsied spleens, tonsils or lymph nodes, or from a peripheral bloodsample. Spleen cells and peripheral blood cells are preferred, theformer because they are a rich source of antibody-producing cells thatare in the dividing plasmablast stage, and the latter because peripheralblood is easily accessible. Often, a panel of animals will have beenimmunized and the spleen of animal with the highest antibody titer willbe removed and the spleen lymphocytes obtained by homogenizing thespleen with a syringe. Typically, a spleen from an immunized mousecontains approximately 5×10⁷ to 2×10⁸ lymphocytes.

The antibody-producing B lymphocytes from the immunized animal are thenfused with cells of an immortal myeloma cell, generally one of the samespecies as the animal that Was immunized Myeloma cell lines suited foruse in hybridoma-producing fusion procedures preferably arenon-antibody-producing, have high fusion efficiency, and enzymedeficiencies that render then incapable of growing in certain selectivemedia which support the growth of only the desired fused cells(hybridomas).

Any one of a number of myeloma cells may be used, as are known to thoseof skill in the art (Goding, 1986; Campbell, 1984). For example, wherethe immunized animal is a mouse, one may use P3-X63 μg8, P3-X63-Ag8.653,NS1/1.Ag 4 1, Sp210-Ag14, FO, NSO/U, MPC-11, MPC11-X45-GTG 1.7 andS194/5XX0 Bul; for rats, one may use R210.RCY3, Y3 Ag 1.2.3, IR983F and4B210; and U-266, GM1500-GRG2, LICR-LON-HMy2 and UC729-6 are all usefulin connection with cell fusions.

Methods for generating hybrids of antibody-producing spleen or lymphnode cells and myeloma cells usually comprise mixing somatic cells withmyeloma cells in a 2:1 ratio, though the ratio may vary from about 20:1to about 1:1, respectively, in the presence of an agent or agents(chemical or electrical) that promote the fusion of cell membranes.Fusion methods using Sendai virus have been described (Kohler andMilstein, 1975; 1976), and those using polyethylene glycol (PEG), suchas 37% (v/v) PEG, by Gefter et al., (1977). The use of electricallyinduced fusion methods is also appropriate (Goding, 1986).

Fusion procedures usually produce viable hybrids at low frequencies,around 1×10⁻⁶ to 1×10⁻⁸. However, this does not pose a problem, as theviable, fused hybrids are differentiated from the parental, unfusedcells (particularly the unfused myeloma cells that would normallycontinue to divide indefinitely) by culturing in a selective medium. Theselective medium is generally one that contains an agent that blocks thede novo synthesis of nucleotides in the tissue culture media. Exemplaryand preferred agents are aminopterin, methotrexate, and azaserine.Aminopterin and methotrexate block de novo synthesis of both purines andpyrimidines, whereas azaserine blocks only purine synthesis. Whereaminopterin or methotrexate is used, the media is supplemented withhypoxanthine and thymidine as a source of nucleotides (HAT medium).Where azaserine is used, the media is supplemented with hypoxanthine.

The preferred selection medium is HAT. Only cells capable of operatingnucleotide salvage pathways are able to survive in HAT medium. Themyeloma cells are defective in key enzymes of the salvage pathway, e.g.,hypoxanthine phosphoribosyl transferase (HPRT), and they cannot survive.The B-cells can operate this pathway, but they have a limited life spanin culture and generally die within about two weeks. Therefore, the onlycells that can survive in the selective media are those hybrids formedfrom myeloma and B-cells.

This culturing provides a population of hybridomas from which specifichybridomas are selected. Typically, selection of hybridomas is performedby culturing the cells by single-clone dilution in microtiter plates,followed by testing the individual clonal supernatants (after about twoto three weeks) for the desired reactivity. The assay should besensitive, simple and rapid, such as radioimmunoassays, enzymeimmunoassays, cytotoxicity assays, plaque assays, dot immunobindingassays, and the like.

The selected hybridomas would then be serially diluted and cloned intoindividual antibody-producing cell lines, which clones can then bepropagated indefinitely to provide mAbs. The cell lines may be exploitedfor mAb production in two basic ways. A sample of the hybridoma can beinjected (often into the peritoneal cavity) into a histocompatibleanimal of the type that was used to provide the somatic and myelomacells for the original fusion. The injected animal develops tumorssecreting the specific monoclonal antibody produced by the fused cellhybrid. The body fluids of the animal, such as serum or ascites fluid,can then be tapped to provide mAbs in high concentration. The individualcell lines could also be cultured in vitro, where the mAbs are naturallysecreted into the culture medium from which they can be readily obtainedin high concentrations. mAbs produced by either means may be furtherpurified, if desired, using filtration, centrifugation and variouschromatographic methods such as HPLC or affinity chromatography.

R. Diagnosing Cancers Involving Fus1, 101F6, Gene 21, Gene 26, Beta*,Luca1, Luca2, PL6, 123F2, or SEM A3

Fus1, 101F6, Gene 21, Gene 26, Beta*, Luca1, Luca2, PL6, 123F2, and SEMA3 and their corresponding genes may be employed as a diagnostic orprognostic indicator of cancer. More specifically, point mutations,deletions, insertions or regulatory perturbations relating to Fus1,101F6, Gene 21, Gene 26, Beta*, Luca1, Luca2, PL6, 123F2, and SEM A3 maycause cancer or promote cancer development, cause or promoter tumorprogression at a primary site, and/or cause or promote metastasis. Otherphenomena associated with malignancy that may be affected by Fus1,101F6, Gene 21, Gene 26, Beta*, Luca1, Luca2, PL6, 123F2, and SEM A3expression include angiogenesis and tissue invasion.

1. Genetic Diagnosis

One embodiment of the instant invention comprises a method for detectingvariation in the expression of Fus1, 101F6, Gene 21, Gene 26, Beta*,Luca1, Luca2, PL6, 123F2, or SEM A3. This may comprise determining thatlevel of Fus1, 101F6, Gene 21, Gene 26, Beta*, Luca1, Luca2, PL6, 123F2,or SEM A3 or determining specific alterations in the expressed product.Obviously, this sort of assay has importance in the diagnosis of relatedcancers. Such cancer may involve cancers of the brain, lung, liver,spleen, kidney, lymph node, small intestine, blood cells, pancreas,colon, stomach, cervix, breast, endometrium, prostate, testicle, ovary,skin, head and neck, esophagus, oral tissue, bone marrow and bloodtissue.

The biological sample can be any tissue or fluid. Various embodimentsinclude cells of the brain, lung, liver, spleen, kidney, lymph node,small intestine, blood cells, pancreas, colon, stomach, cervix, breast,endometrium, prostate, testicle, ovary, skin, head and neck, esophagus,oral tissue, bone marrow and blood tissue. Other embodiments includefluid samples such as peripheral blood, lymph fluid, ascites, serousfluid, pleural effusion, sputum, cerebrospinal fluid, lacrimal fluid,stool, or urine.

Nucleic acid used is isolated from cells contained in the biologicalsample, according to standard methodologies (Sambrook et al., 1989). Thenucleic acid may be genomic DNA or fractionated or whole cell RNA. WhereRNA is used, it may be desired to convert the RNA to a complementaryDNA. In one embodiment, the RNA is whole cell RNA; in another, it ispoly-A RNA. Normally, the nucleic acid is amplified.

Depending on the format, the specific nucleic acid of interest isidentified in the sample directly using amplification or with a second,known nucleic acid following amplification. Next, the identified productis detected. In certain applications, the detection may be performed byvisual means (e.g., ethidium bromide staining of a gel). Alternatively,the detection may involve indirect identification of the product viachemiluminescence, radioactive scintigraphy of radiolabel or fluorescentlabel or even via a system using electrical or thermal impulse signals(Affymax Technology; Bellus, 1994).

Following detection, one may compare the results seen in a given patientwith a statistically significant reference group of normal patients andpatients that have Fus1, 101F6, Gene 21, Gene 26, Beta*, Luca1, Luca2,PL6, 123F2, or SEM A3-related pathologies. In this way, it is possibleto correlate the amount or kind of Fus1, 101F6, Gene 21, Gene 26, Beta*,Luca1, Luca2, PL6, 123F2, or SEM A3 detected with various clinicalstates.

Alterations of a gene include deletions, insertions, point mutations andduplications. Point mutations result in stop codons, frameshiftmutations or amino acid substitutions. Somatic mutations are thoseoccurring in non-germline tissues. Germ-line tissue can occur in anytissue and are inherited. Mutations in and outside the coding regionalso may affect the amount of Fus1, 101F6, Gene 21, Gene 26, Beta*,Luca1, Luca2, PL6, 123F2, or SEM A3 produced, both by altering thetranscription of the gene or in destabilizing or otherwise altering theprocessing of either the transcript (mRNA) or protein.

A variety of different assays are contemplated in this regard, includingbut not limited to, fluorescent in situ hybridization (FISH), direct DNAsequencing, PFGE analysis, Southern or Northern blotting,single-stranded conformation analysis (SSCA), RNAse protection assay,allele-specific oligonucleotide (ASO), dot blot analysis, denaturinggradient gel electrophoresis, RFLP and PCRT™-SSCP.

2. Southern/Northern Blotting

Blotting techniques are well known to those of skill in the art.Southern blotting involves the use of DNA as a target, whereas Northernblotting involves the use of RNA as a target. Each provide differenttypes of information, although cDNA blotting is analogous, in manyaspects, to blotting or RNA species.

Briefly, a probe is used to target a DNA or RNA species that has beenimmobilized on a suitable matrix, often a filter of nitrocellulose. Thedifferent species should be spatially separated to facilitate analysis.This often is accomplished by gel electrophoresis of nucleic acidspecies followed by “blotting” on to the filter.

Subsequently, the blotted target is incubated with a probe (usuallylabeled) under conditions that promote denaturation and rehybridization.Because the probe is designed to base pair with the target, the probewill binding a portion of the target sequence under renaturingconditions. Unbound probe is then removed, and detection is accomplishedas described above.

3. Separation Methods

It normally is desirable, at one stage or another, to separate theamplification product from the template and the excess primer for thepurpose of determining whether specific amplification has occurred. Inone embodiment, amplification products are separated by agarose,agarose-acrylamide or polyacrylamide gel electrophoresis using standardmethods. See Sambrook et al., 1989.

Alternatively, chromatographic techniques may be employed to effectseparation. There are many kinds of chromatography which may be used inthe present invention: adsorption, partition, ion-exchange and molecularsieve, and many specialized techniques for using them including column,paper, thin-layer and gas chromatography (Freifelder, 1982).

4. Detection Methods

Products may be visualized in order to confirm amplification of themarker sequences. One typical visualization method involves staining ofa gel with ethidium bromide and visualization under UV light.Alternatively, if the amplification products are integrally labeled withradio- or fluorometrically-labeled nucleotides, the amplificationproducts can then be exposed to x-ray film or visualized under theappropriate stimulating spectra, following separation.

5. Kit Components

All the essential materials and reagents required for detecting andsequencing Fus1, 101F6, Gene 21, Gene 26, Beta*, Luca1, Luca2, PL6,123F2, or SEM A3 and variants thereof may be assembled together in akit. This generally will comprise preselected primers and probes. Alsoincluded may be enzymes suitable for amplifying nucleic acids includingvarious polymerases (RT, Taq, Sequenase™, etc.), deoxynucleotides, andbuffers to provide the necessary reaction mixture for amplification.Such kits also generally will comprise, in suitable means, distinctcontainers for each individual reagent and enzyme as well as for eachprimer or probe.

6. RT-PCR™ (Relative Quantitative)

Reverse transcription (RT) of RNA to cDNA followed by relativequantitative PCR™ (RT-PCR™) can be used to determine the relativeconcentrations of specific mRNA species isolated from patients. Bydetermining that the concentration of a specific mRNA species varies, itis shown that the gene encoding the specific mRNA species isdifferentially expressed.

In PCR™, the number of molecules of the amplified target DNA increase bya factor approaching two with every cycle of the reaction until somereagent becomes limiting. Thereafter, the rate of amplification becomesincreasingly diminished until there is no increase in the amplifiedtarget between cycles. If a graph is plotted in which the cycle numberis on the X axis and the log of the concentration of the amplifiedtarget DNA is on the Y axis, a curved line of characteristic shape isformed by connecting the plotted points. Beginning with the first cycle,the slope of the line is positive and constant. This is said to be thelinear portion of the curve. After a reagent becomes limiting, the slopeof the line begins to decrease and eventually becomes zero. At thispoint the concentration of the amplified target DNA becomes asymptoticto some fixed value. This is said to be the plateau portion of thecurve.

The concentration of the target DNA in the linear portion of the PCR™amplification is directly proportional to the starting concentration ofthe target before the reaction began. By determining the concentrationof the amplified products of the target DNA in PCR™ reactions that havecompleted the same number of cycles and are in their linear ranges, itis possible to determine the relative concentrations of the specifictarget sequence in the original DNA mixture. If the DNA mixtures arecDNAs synthesized from RNAs isolated from different tissues or cells,the relative abundances of the specific mRNA from which the targetsequence was derived can be determined for the respective tissues orcells. This direct proportionality between the concentration of the PCR™products and the relative mRNA abundances is only true in the linearrange of the PCR™ reaction.

The final concentration of the target DNA in the plateau portion of thecurve is determined by the availability of reagents in the reaction mixand is independent of the original concentration of target DNA,Therefore, the first condition that must be met before the relativeabundances of a mRNA species can be determined by RT-PCR™ for acollection of RNA populations is that the concentrations of theamplified PCR™ products must be sampled when the PCR™ reactions are inthe linear portion of their curves.

The second condition that must be met for an RT-PCR™ experiment tosuccessfully determine the relative abundances of a particular mRNAspecies is that relative concentrations of the amplifiable cDNAs must benormalized to some independent standard. The goal of an RT-PCR™experiment is to determine the abundance of a particular mRNA speciesrelative to the average abundance of all mRNA species in the sample. Inthe experiments described below, mRNAs for β-actin, asparaginesynthetase and lipocortin II were used as external and internalstandards to which the relative abundance of other mRNAs are compared.

Most protocols for competitive PCR™ utilize internal PCR™ standards thatare approximately as abundant as the target. These strategies areeffective if the products of the PCR™ amplifications are sampled duringtheir linear phases. If the products are sampled when the reactions areapproaching the plateau phase, then the less abundant product becomesrelatively over represented. Comparisons of relative abundancies madefor many different RNA samples, such as is the case when examining RNAsamples for differential expression, become distorted in such a way asto make differences in relative abundances of RNAs appear less than theyactually are. This is not a significant problem if the internal standardis much more abundant than the target. If the internal standard is moreabundant than the target, then direct linear comparisons can be madebetween RNA samples.

The above discussion describes theoretical considerations for an RT-PCR™assay for clinically derived materials. The problems inherent inclinical samples are that they are of variable quantity (makingnormalization problematic), and that they are of variable quality(necessitating the co-amplification of a reliable internal control,preferably of larger size than the target). Both of these problems areovercome if the RT-PCR™ is performed as a relative quantitative RT-PCR™with an internal standard in which the internal standard is anamplifiable cDNA fragment that is larger than the target cDNA fragmentand in which the abundance of the mRNA encoding the internal standard isroughly 5-100 fold higher than the mRNA encoding the target. This assaymeasures relative abundance, not absolute abundance of the respectivemRNA species.

Other studies may be performed using a more conventional relativequantitative RT-PCR™ assay with an external standard protocol. Theseassays sample the PCR™ products in the linear portion of theiramplification curves. The number of PCR™ cycles that are optimal forsampling must be empirically determined for each target cDNA fragment.In addition, the reverse transcriptase products of each RNA populationisolated from the various tissue samples must be carefully normalizedfor equal concentrations of amplifiable cDNAs. This consideration isvery important since the assay measures absolute mRNA abundance.Absolute mRNA abundance can be used as a measure of differential geneexpression only in normalized samples. While empirical determination ofthe linear range of the amplification curve and normalization of cDNApreparations are tedious and time consuming processes, the resultingRT-PCR™ assays can be superior to those derived from the relativequantitative RT-PCR™ assay with an internal standard.

One reason for this advantage is that without the internalstandard/competitor, all of the reagents can be converted into a singlePCR™ product in the linear range of the amplification curve, thusincreasing the sensitivity of the assay. Another reason is that withonly one PCR™ product, display of the product on an electrophoretic gelor another display method becomes less complex, has less background andis easier to interpret.

Still other studies may be performed using “real-time” RT-PCR™ (Higuchiet al., 1993). These assays detect PCR™ products as they accumulateinstead of detecting the amount of PCR™ products accumulated after afixed number of cycles. A method of detecting fluorescence after eachPCR™ cycle is required. The fluorescence signal is plotted versus thecycle number. The cycle number is expressed as the threshold cycle(C_(T)). The initial fluorescence defines the baseline for the plot andan accumulated PCR™ product is indicated by an increase in fluorescenceabove the baseline. Quantification of the amount of target in a sampleis determined by measuring and comparing the C_(T) to a standard curveto determine the starting copy number.

“Real-Time” RT-PCR™ (Higuchi et al., 1993) provides more precisequantitation of the amount of target because it is determined during theexponential phase of PCR™, rather than at the endpoint. It also allowshigher throughput because the use of C_(T) values allow a larger dynamicrange. Dilutions of each sample are no longer required.

7. Immunodiagnosis

Antibodies of the present invention can be used in characterizing theFus1, 101F6, Gene 21, Gene 26, Beta*, Luca1, Luca2, PL6, 123F2, or SEMA3 content of healthy and diseased tissues, through techniques such asELISAs and Western blotting. This may provide a screen for the presenceor absence of malignancy or as a predictor of future cancer.

The use of antibodies of the present invention, in an ELISA assay iscontemplated. For example, anti-Fus1, 101F6, Gene 21, Gene 26, Beta*,Luca1, Luca2, PL6, 123F2, or SEM A3 antibodies are immobilized onto aselected surface, preferably a surface exhibiting a protein affinitysuch as the wells of a polystyrene microtiter plate. After washing toremove incompletely adsorbed material, it is desirable to bind or coatthe assay plate wells with a non-specific protein that is known to beantigenically neutral with regard to the test antisera such as bovineserum albumin (BSA), casein or solutions of powdered milk. This allowsfor blocking of non-specific adsorption sites on the immobilizingsurface and thus reduces the background caused by non-specific bindingof antigen onto the surface.

After binding of antibody to the well, coating with a non-reactivematerial to reduce background, and washing to remove unbound material,the immobilizing surface is contacted with the sample to be tested in amanner conducive to immune complex (antigen/antibody) formation.

Following formation of specific immunocomplexes between the test sampleand the bound antibody, and subsequent washing, the occurrence and evenamount of immunocomplex formation may be determined by subjecting sameto a second antibody having specificity for Fus1, 101F6, Gene 21, Gene26, Beta*, Luca1, Luca2, PL6, 123F2, or SEM A3 that differs the firstantibody. Appropriate conditions preferably include diluting the samplewith diluents such as BSA, bovine gamma globulin (BGG) and phosphatebuffered saline (PBS)/Tween®. These added agents also, tend to assist inthe reduction of nonspecific background. The layered antisera is thenallowed to incubate for from about 2 to about 4 hr, at temperaturespreferably on the order of about 25° to about 27° C. Followingincubation, the antisera-contacted surface is washed so as to removenon-immunocomplexed material. A preferred washing procedure includeswashing with a solution such as PBS/Tween®, or borate buffer.

To provide a detecting means, the second antibody will preferably havean associated enzyme that will generate a color development uponincubating with an appropriate chromogenic substrate. Thus, for example,one will desire to contact and incubate the second antibody-boundsurface with a urease, alkaline phosphatase, glucose oxidase, or(horseradish) peroxidase-conjugated anti-human IgG for a period of timeand under conditions which favor the development of immunocomplexformation (e.g., incubation for 2 hr at room temperature in aPBS-containing solution such as PBS/Tween®).

After incubation with the second enzyme-tagged antibody, and subsequentto washing to remove unbound material, the amount of label is quantifiedby incubation with a chromogenic substrate such as urea and bromocresolpurple or 2,2′-azino-di-(3-ethyl-benzthiazoline)-6-sulfonic acid (ABTS)and H2O2, in the case of peroxidase as the enzyme label. Quantitation isthen achieved by measuring the degree of color generation, e.g., using avisible spectrum spectrophotometer.

The preceding format may be altered by first binding the sample to theassay plate. Then, primary antibody is incubated with the assay plate,followed by detecting of bound primary antibody using a labeled secondantibody with specificity for the primary antibody.

The antibody compositions of the present invention will find great usein immunoblot or Western blot analysis. The antibodies may be used ashigh-affinity primary reagents for the identification of proteinsimmobilized onto a solid support matrix, such as nitrocellulose, nylonor combinations thereof. In conjunction with immunoprecipitation,followed by gel electrophoresis, these may be used as a single stepreagent for use in detecting antigens against which secondary reagentsused in the detection of the antigen cause an adverse background.Immunologically-based detection methods for use in conjunction withWestern blotting include enzymatically-, radiolabel-, orfluorescently-tagged secondary antibodies against the toxin moiety areconsidered to be of particular use in this regard.

8. Combination of Tumor Suppressors with Other Markers

Tumors are notoriously heterogeneous, particularly in advanced stages oftumor progression (Morton et al., 1993; Fidler and Hart, 1982; Nowell,1982; Elder et al., 1989; Bystryn et al., 1985). Although tumor cellswithin a primary tumor or metastasis all may express the same markergene, the level of specific mRNA expression can vary considerably (Elderet al., 1989). It is, in certain instances, necessary to employ adetection system that can cope with an array of heterogeneous markers.

Thus, while the present invention exemplifies various tumor suppressorsas a markers, any marker that is correlated with the presence or absenceof cancer may be used in combination with these markers to improve theefficacy of tumor detection and treatment. A marker, as used herein, isany proteinaceous molecule (or corresponding gene) whose production orlack of production is characteristic of a cancer cell. Depending on theparticular set of markers employed in a given analysis, the statisticalanalysis will vary. For example, where a particular combination ofmarkers is highly specific for melanomas or breast cancer, thestatistical significance of a positive result will be high. It may be,however, that such specificity is achieved at the cost of sensitivity,i.e., a negative result may occur even in the presence of melanoma orbreast cancer. By the same token, a different combination may be verysensitive, i.e., few false negatives, but has a lower specificity.

As new markers are identified, different combinations may be developedthat show optimal function with different ethnic groups or sex,different geographic distributions, different stages of disease,different degrees of specificity or different degrees of sensitivity . .. . Marker combinations also may be developed, which are particularlysensitive to the effect of therapeutic regimens on disease progression.Patients may be monitored after surgery, gene therapy, hyperthermia,immunotherapy, cytokine therapy, gene therapy, radiotherapy orchemotherapy, to determine if a specific therapy is effective.

One particularly useful combination of markers for melanoma istyrosinase and members of the MAGE family, particularly MAGE-1 orMAGE-3. Human tyrosinase is an essential enzyme which regulates theproduction of melanin (Nordlund et al., 1989; Hoon et al., 1993), agroup of brown or black pigments in the skin and eyes of humans. Morespecifically, tyrosinase catalyzes the conversion of tyrosine to Dopaand of Dopa to dopaquinone.

There are many other markers that may be used in combination with these,and other, markers. For example, b-human chorionic gonadotropin (b-HCG).b-HCG is produced by trophoblastic cells of placenta of pregnant womanand is essential for maintenance of pregnancy at the early stages(Pierce et al., 1981; Talmadge et al., 1984). b-HCG is known to beproduced by trophoblastic or germ cell origin tumors, such aschoriocarcinoma or testicular carcinoma cells (Madersbacher et al.,1994; Cole et al., 1983). Also ectopic expression of b-HCG has beendetected by a number of different immunoassays in various tumors ofnon-gonadal such as breast, lung, gastric, colon, and pancreas, etc.(McManus et al., 1976; Yoshimura et al., 1994; Yamaguchi et al., 1989;Marcillac et al., 1992; Alfthan et al., 1992). Although the function ofb-HCG production in these tumors is still unknown, the atavisticexpression of b-HCG by cancer cells and not by normal cells ofnon-gonadal origin suggests it may be a potentially good marker in thedetection of melanoma and breast cancer (Hoon et al., 1996; Sarantou etal., 1997).

Another exemplary example of a marker is glycosyltransferaseb-1,4-N-acetylgalacto-saminyltransferase (GalNAc). GalNAc catalyzes thetransfer of N-acetylgalactosamine by b1(r) 4 linkage onto bothgangliosides GM3 and GD3 to generate GM2 and GD2, respectively (Nagataet al., 1992; Furukawa et al., 1993). It also catalyzes the transfer ofN-acetylgalactosamine to other carbohydrate molecules such as mucins.Gangliosides are glycosphingolipids containing sialic acids which playan important role in cell differentiation, adhesion and malignanttransformation. In melanoma, gangliosides GM2 and GD2 expression, areoften enhanced to very high levels and associate with tumor progressionincluding metastatic tumors (Hoon et al., 1989; Ando et al., 1987;Cambia et al., 1984; Tsuchida et al., 1987a). Gangliosides are alsoexpressed in melanoma, renal, lung, breast carcinoma cancer cells. Thegangliosides GM2 and GD2 are immunogenic in humans and can be used as atarget for specific immunotherapy such as human monoclonal antibodies orcancer vaccines (Tsuchida et al., 1987b; hie, 1985.)

GalNAc mRNA may be used as a marker of GM2 and GD2 expression andconsequently a marker of either melanoma or breast cancer cells. GalNAcis generally not expressed in normal lymphocytes, epithelial cells,melanocytes, connective tissue or lymph node cells. If detected, it isin very low levels. Prostate specific antigen is a well characterizedmarker for prostate cancer (Gomella et al., 1997). bcr/abl gene forleukemia is a further well characterized marker that is contemplated tobe useful in combination with HOJ-1.

Other markers contemplated by the present invention include cytolytic Tlymphocyte (CTL) targets. MAGE-3 is a marker identified in melanomacells and breast carcinoma. MAGE-3 is expressed in many melanomas aswell as other tumors and is a (CTL) target (Gaugler et al., 1994).MAGE-1, MAGE-2, MAGE-4, MAGE-6, MAGE-12, MAGE-Xp, and are other membersof the MAGE gene family. MAGE-1 gene sequence shows 73% identity withMAGE-3 and expresses an antigen also recognized by CTL (Gaugler et al.,1994). MART-1 is another potential CTL target (Robbins et al., 1994) andalso may be included in the present invention.

MUC18 is another marker that is useful in the identification of melanomacells (Lehman et al., 1989; Lehman et al., 1987). MUC18 is a cellsurface glycoprotein that is a member of the immunoglobulin superfamilyand possesses sequence homology to neural cell adhesion molecules(NCAM). Other mucin family members include MUC1, MUC2, MUC3 and MUC4.These were found to be expressed at a high level in certain tumor celllines (Hollingsworth et al., 1994) and also may be used as markers incombination with the novel H03-1 marker of the present invention.

Other members of the immunoglobulin superfamily of adhesion moleculesassociated with the development of melanoma metastasis (Denton et al.,1992) may be utilized in the present invention. Examples includeintercellular adhesion molecule-1 (ICAM-1) NCAM, VCAM-1 and ELAM.Another embodiment includes carcinoma cell related molecules andmolecules associated with other metastatic diseases such ascarcinoembryonic antigen (CEA; Lin and Guidotti, 1989).

Other carcinoma or skin cancer associated proteins and theircorresponding nucleic acids also may be utilized in the presentinvention. Preferred examples include melanoma antigen gp75(Vijayasardahi et al., 1990), human cytokeratin 20, high molecularweight melanoma antigen (Natali et al., 1987) and cytokeratin 19 (K19)(Datta et al., 1994). Other markers that may be useful herein includeinhibitors of the cyclin-dependent kinases, (CDK). For example, CDK4regulates progression through the G1 phase of the cell cycle. Theactivity of CDK4 is controlled by an activating subunit, D-type cyclin,and by an inhibitory subunit, the p16INK4 has been biochemicallycharacterized as a protein that specifically binds to and inhibits CDK4(Serrano et al., 1993; Serrano et al., 1995). Other CDK-inhibitoryproteins that also includes p16, p21WAF1, and p27KIP1. This list is notintended to be exhaustive, but merely exemplary, for the type and numberof potential markers which may be used in the present invention.

Other proteins and their corresponding nucleic acids related to themelanin synthesis pathway may be used as markers, such as tyrosinaserelated protein 1 and 2 and members of the pMel 17 gene family (Kwon etal., 1993).

Preferred embodiments of the invention involve many differentcombinations of markers for the detection of cancer cells. Any markerthat is indicative of neoplasia in cells may be included in thisinvention.

S. Transgenic Animals/Knockout Animals

In one embodiment of the invention, transgenic animals are producedwhich contain a functional transgene encoding a functional Fus1, 101F6,Gene 21, Gene 26, Beta*, Luca1, Luca2, PL6, 123F2, or SEM A3 polypeptideor variants thereof. Transgenic animals expressing Fus1, 101F6, Gene 21,Gene 26, Beta*, Luca1, Luca2, PL6, 123F2, or SEM A3 transgenes,recombinant cell lines derived from such animals and transgenic embryosmay be useful in methods for screening for and identifying agents thatinduce or repress function of Fus1, 101F6, Gene 21, Gene 26, Beta*,Luca1, Luca2, PL6, 123F2, or SEM A3. Transgenic animals of the presentinvention also can be used as models for studying indications such ascancers.

In one embodiment of the invention, a Fus1, 101F6, Gene 21, Gene 26,Beta*, Luca1, Luca2, PL6, 123F2, or SEM A3 transgene is introduced intoa non-human host to produce a transgenic animal expressing a human ormurine Fus1, 101F6, Gene 21, Gene 26, Beta*, Luca1, Luca2, PL6, 123F2,or SEM A3 gene. The transgenic animal is produced by the integration ofthe transgene into the genome in a manner that permits the expression ofthe transgene. Methods for producing transgenic animals are generallydescribed by Wagner and Hoppe (U.S. Pat. No. 4,873,191; which isincorporated herein by reference), Brinster et al. 1985; which isincorporated herein by reference in its entirety) and in “Manipulatingthe Mouse Embryo; A Laboratory Manual” 2nd edition (eds., Hogan,Beddington, Costantimi and Long, Cold Spring Harbor Laboratory Press,1994; which is incorporated herein by reference in its entirety).

It may be desirable to replace the endogenous Fus1, 101F6, Gene 21, Gene26, Beta*, Luca1, Luca2, PL6, 123F2, or SEM A3 by homologousrecombination between the transgene and the endogenous gene; or theendogenous gene may be eliminated by deletion as in the preparation of“knock-out” animals. Typically, a Fus1, 101F6, Gene 21, Gene 26, Beta*,Luca1, Luca2, PL6, 123F2, or SEM A3 gene flanked by genomic sequences istransferred by microinjection into a fertilized egg. The microinjectedeggs are implanted into a host female, and the progeny are screened forthe expression of the transgene. Transgenic animals may be produced fromthe fertilized eggs from a number of animals including, but not limitedto reptiles, amphibians, birds, mammals, and fish. Within a particularembodiment, transgenic mice are generated which overexpress Fus1, 101F6,Gene 21, Gene 26, Beta*, Luca1, Luca2, PL6, 123F2, or SEM A3 or expressa mutant form of the polypeptide. Alternatively, the absence of a Fus1,101F6, Gene 21, Gene 26, Beta*, Luca1, Luca2, PL6, 123F2, or SEM A3 in“knock-out” mice permits the study of the effects that loss of Fus1,101F6, Gene 21, Gene 26, Beta*, Luca1, Luca2, PL6, 123F2, or SEM A3protein has on a cell in vivo. Knock-out mice also provide a model forthe development of Fus1, 101F6, Gene 21, Gene 26, Beta*, Luca1, Luca2,PL6, 123F2, or SEM A3-related cancers.

As noted above, transgenic animals and cell lines derived from suchanimals may find use in certain testing experiments. In this regard,transgenic animals and cell lines capable of expressing wild-type ormutant Fus1, 101F6, Gene 21, Gene 26, Beta*, Luca1, Luca2, PL6, 123F2,or SEM A3 may be exposed to test substances. These test substances canbe screened for the ability to enhance wild-type Fus1, 101F6, Gene 21,Gene 26, Beta*, Luca1, Luca2, PL6, 123F2, or SEM A3 expression and orfunction or impair the expression or function of mutant Fus1, 101F6,Gene 21, Gene 26, Beta*, Luca1, Luca2, PL6, 123F2, or SEM A3.

Promoter sequences mentioned within this document may be used to driveβ-galactosidase expression. The use of a β-galactosidase reporterconstruct in transgenic mice may be used to identify factors whichregulate Fus1, 101F6, Gene 21, Gene 26, Beta*, Luca1, Luca2, PL6, 123F2,or SEM A3 expression.

T. Methods for Treating Cancers Using Fus1, 101F6, Gene 21, Gene 26,Beta*, Luca1, Luca2, PL6, 123F2, or SEM A3

The present invention also involves, in another embodiment, thetreatment of cancer. The types of cancer that may be treated, accordingto the present invention, is limited only by the involvement of Fus1,101F6, Gene 21, Gene 26, Beta*, Luca1, Luca2, PL6, 123F2, or SEM A3. Byinvolvement, it is not even a requirement that Fus1, 101F6, Gene 21,Gene 26, Beta*, Luca1, Luca2, PL6, 123F2, or SEM A3 be mutated orabnormal—the overexpression of Fus1, 101F6, Gene 21, Gene 26, Beta*,Luca1, Luca2, PL6, 123F2, or SEM A3 may actually overcome other lesionswithin the cell. Thus, it is contemplated that a wide variety of cancercells may be treated using Fus1, 101F6, Gene 21, Gene 26, Beta*, Luca1,Luca2, PL6, 123F2, or SEM A3 therapy, including brain, lung, liver,spleen, kidney, lymph node, small intestine, blood cells, pancreas,colon, stomach, cervix, breast, endometrium, prostate, testicle, ovary,skin, head and neck, esophagus, oral tissue, bone marrow and bloodtissue.

In many contexts, it is not necessary that the cancer cell be killed orinduced to undergo normal cell death or “apoptosis.” Rather, toaccomplish a meaningful treatment, all that is required is that thetumor growth be slowed to some degree. It may be that the tumor growthis partially or completely blocked, however, or that some tumorregression is achieved. Clinical terminology such as “remission” and“reduction of tumor” burden also are contemplated given their normalusage.

1. Genetic Based Therapies

One of the therapeutic embodiments contemplated by the present inventorsis the intervention, at the molecular level, in the events involved inthe tumorigenesis of some cancers. Specifically, the present inventorsintend to provide, to a cancer cell, an expression cassette capable ofproviding Fus1, 101F6, Gene 21, Gene 26, Beta*, Luca1, Luca2, PL6,123F2, or SEM A3 to that cell. The lengthy discussion of expressionvectors and the genetic elements employed therein is incorporated intothis section by reference. Particularly preferred expression vectors areviral vectors such as adenovirus, adeno-associated virus, herpesvirus,vaccinia virus and retrovirus. Also preferred isliposomally-encapsulated expression vector.

Various routes are contemplated for various tumor types. The sectionbelow on routes contains an extensive list of possible routes. Forpractically any tumor, systemic delivery is contemplated. This willprove especially important for attacking microscopic or metastaticcancer. Where discrete tumor mass may be identified, a variety ofdirect, local and regional approaches may be taken. For example, thetumor may be directly injected with the expression vector. A tumor bedmay be treated prior to, during or after resection. Following resection,one generally will deliver the vector by a catheter left in placefollowing surgery. One may utilize the tumor vasculature to introducethe vector into the tumor by injecting a supporting vein or artery. Amore distal blood supply route also may be utilized.

In a different embodiment, ex vivo gene therapy is contemplated. Thisapproach is particularly suited, although not limited, to treatment ofbone marrow associated cancers. In an ex vivo embodiment, cells from thepatient are removed and maintained outside the body for at least someperiod of time. During this period, a therapy is delivered, after whichthe cells are reintroduced into the patient; hopefully, any tumor cellsin the sample have been killed.

2. Protein Therapy

Another therapy approach is the provision, to a subject, of Fus1, 101F6,Gene 21, Gene 26, Beta*, Luca1, Luca2, PL6, 123F2, or SEM A3polypeptide, active fragments, synthetic peptides, mimetics or otheranalogs thereof. The protein may be produced by recombinant expressionmeans or, if small enough, generated by an automated peptidesynthesizer. Formulations would be selected based on the route ofadministration and purpose including, but not limited to, liposomalformulations and classic pharmaceutical preparations.

3. Combined Therapy with Immunotherapy, Traditional Chemo- orRadiotherapy

Tumor cell resistance to DNA damaging agents represents a major problemin clinical oncology. One goal of current cancer research is to findways to improve the efficacy of chemo- and radiotherapy. One way is bycombining such traditional therapies with gene therapy. For example, theherpes simplex-thymidine kinase (HS-tk) gene, when delivered to braintumors by a retroviral vector system, successfully inducedsusceptibility to the antiviral agent ganciclovir (Culver et al., 1992).In the context of the present invention, it is contemplated that Fus1,101F6, Gene 21, Gene 26, Beta*, Luca1, Luca2, PL6, 123F2, or SEM A3replacement therapy could be used similarly in conjunction with chemo-or radiotherapeutic intervention. It also may prove effective to combineFus1, 101F6, Gene 21, Gene 26, Beta*, Luca1, Luca2, PL6, 123F2, or SEMA3 gene therapy with immunotherapy, as described above.

To kill cells, inhibit cell growth, inhibit metastasis, inhibitangiogenesis or otherwise reverse or reduce the malignant phenotype oftumor cells, using the methods and compositions of the presentinvention, one would generally contact a “target” cell with a Fus1,101F6, Gene 21, Gene 26, Beta*, Luca1, Luca2, PL6, 123F2, or SEM A3expression construct and at least one other agent. These compositionswould be provided in a combined amount effective to kill or inhibitproliferation of the cell. This process may involve contacting the cellswith the expression construct and the agent(s) or factor(s) at the sametime. This may be achieved by contacting the cell with a singlecomposition or pharmacological formulation that includes both agents, orby contacting the cell with two distinct Compositions or formulations,at the same time, wherein one composition includes the expressionconstruct and the other includes the agent.

Alternatively, the gene therapy treatment may precede or follow theother agent treatment by intervals ranging from minutes to weeks. Inembodiments where the other agent and expression construct are appliedseparately to the cell, one would generally ensure that a significantperiod of time did not expire between the time of each delivery, suchthat the agent and expression construct would still be able to exert anadvantageously combined effect on the cell. In such instances, it iscontemplated that one would contact the cell with both modalities withinabout 12-24 hours of each other and, more preferably, within about 6-12hours of each other, with a delay time of only about 12 hours being mostpreferred. In some situations, it may be desirable to extend the timeperiod for treatment significantly, however, where several days (2, 3,4, 5, 6 or 7) to several weeks (1, 2, 3, 4, 5, 6, 7 or 8) lapse betweenthe respective administrations.

It also is conceivable that more than one administration of either Fus1,101F6, Gene 21, Gene 26, Beta*, Luca1, Luca2, PL6, 123F2, or SEM A3 orthe other agent will be desired. Various combinations may be employed,where Fus1, 101F6, Gene 21, Gene 26, Beta*, Luca1, Luca2, PL6, 123F2, orSEM A3 is “A” and the other agent is “B”, as exemplified below:

A/B/A B/A/B B/B/A A/A/B B/A/A A/B/B B/B/B/A B/B/A/B A/A/B/B A/B/A/BA/B/B/A B/B/A/A B/A/B/A B/A/A/B B/B/B/A A/A/A/B B/A/A/A A/B/A/A A/A/B/AA/B/B/B B/A/B/B B/B/A/BOther combinations are contemplated. Again, to achieve cell killing,both agents are delivered to a cell in a combined amount effective tokill the cell.

Agents or factors suitable for use in a combined therapy are anychemical compound or treatment method that induces DNA damage whenapplied to a cell. Such agents and factors include radiation and wavesthat induce DNA damage such as, γ-irradiation, X-rays, acceleratedprotons, UV-irradiation, microwaves, electronic emissions, and the like.A variety of chemical compounds, also described as “chemotherapeuticagents,” function to induce DNA damage, all of which are intended to beof use in the combined treatment methods disclosed herein.Chemotherapeutic agents contemplated to be of use, include, e.g.,cisplatin (CDDP), carboplatin, procarbazine, mechlorethamine,cyclophosphamide, camptothecin, ifosfamide, melphalan, chlorambucil,busulfan, nitrosurea, dactinomycin, daunorubicin, doxorubicin,bleomycin, plicomycin, mitomycin, etoposide (VP16), tamoxifen,raloxifene, estrogen receptor binding agents, taxol, gemcitabien,navelbine, farnesyl-protein tansferase inhibitors, transplatinum,5-fluorouracil, vincristin, vinblastin and methotrexate and evenhydrogen peroxide. The invention also encompasses the use of acombination of one or more DNA damaging agents, whether radiation-basedor actual compounds, such as the use of X-rays with cisplatin or the useof cisplatin with etoposide. In certain embodiments, the use ofcisplatin in combination with a Fus1, 101F6, Gene 21, Gene 26, Beta*,Luca1, Luca2, PL6, 123F2, or SEM A3 expression construct is particularlypreferred as this compound.

In treating cancer according to the invention, one would contact thetumor cells with an agent in addition to the expression construct. Thismay be achieved by irradiating the localized tumor site with radiationsuch as X-rays, accelerated protons, UV-light, γ-rays or evenmicrowaves. Alternatively, the tumor cells may be contacted with theagent by administering to the subject a therapeutically effective amountof a pharmaceutical composition comprising a compound such as,adriamycin, 5-fluorouracil, etoposide, camptothecin, actinomycin-D,mitomycin C, or more preferably, cisplatin. The agent may be preparedand used as a combined therapeutic composition, or kit, by combining itwith a Fus1, 101F6, Gene 21, Gene 26, Beta*, Luca1, Luca2, PL6, 123F2,or SEM A3 expression construct, as described above.

Agents that directly cross-link nucleic acids, specifically DNA, areenvisaged to facilitate DNA damage leading to a synergistic,antineoplastic combination with Fus1, 101F6, Gene 21, Gene 26, Beta*,Luca1, Luca2, PL6, 123F2, or SEM A3. Agents such as cisplatin, and otherDNA alkylating agents may be used. Cisplatin has been widely used totreat cancer, with efficacious doses used in clinical applications of 20mg/m² for 5 days every three weeks for a total of three courses.Cisplatin is not absorbed orally and must therefore be delivered viainjection intravenously, subcutaneously, intratumorally orintraperitoneally.

Agents that damage DNA also include compounds that interfere with DNAreplication, mitosis and chromosomal segregation. Such chemotherapeuticcompounds include adriamycin, also known as doxorubicin, etoposide,verapamil, podophyllotoxin, and the like. Widely used in a clinicalsetting for the treatment of neoplasms, these compounds are administeredthrough bolus injections intravenously at doses ranging from 25-75 mg/m²at 21 day intervals for adriamycin, to 35-50 mg/m² for etoposideintravenously or double the intravenous dose orally.

Agents that disrupt the synthesis and fidelity of nucleic acidprecursors and subunits also lead to DNA damage. As such a number ofnucleic acid precursors have been developed. Particularly useful areagents that have undergone extensive testing and are readily available.As such, agents such as 5-fluorouracil (5-FU), are preferentially usedby neoplastic tissue, making this agent particularly useful fortargeting to neoplastic cells. Although quite toxic, 5-FU, is applicablein a wide range of carriers, including topical, however intravenousadministration with doses ranging from 3 to 15 mg/kg/day being commonlyused.

Other factors that cause DNA damage and have been used extensivelyinclude what are commonly known as γ-rays; X-rays, accelerated protons,and/or the directed delivery of radioisotopes to tumor cells. Otherforms of DNA damaging factors are also contemplated such as microwaves,and UV-irradiation. It is most likely that all of these factors effect abroad range of damage DNA, on the precursors of DNA, the replication andrepair of DNA, and the assembly and maintenance of chromosomes. Dosageranges for X-rays range from daily doses of 50 to 200 roentgens forprolonged periods of time (3 to 4 weeks), to single doses of 2000 to6000 roentgens. Dosage ranges for radioisotopes vary widely, and dependon the half-life of the isotope, the strength and type of radiationemitted, and the uptake by the neoplastic cells.

The skilled artisan is directed to “Remington's Pharmaceutical Sciences”15th Edition, chapter 33, in particular pages 624-652. Some variation indosage will necessarily occur depending on the condition of the subjectbeing treated. The person responsible for administration will, in anyevent, determine the appropriate dose for the individual subject.Moreover, for human administration, preparations should meet sterility,pyrogenicity, general safety and purity standards as required by FDAOffice of Biologics Standards.

The inventors propose that the regional delivery of Fus1, 101F6, Gene21, Gene 26, Beta*, Luca1, Luca2, PL6, 123F2, or SEM A3 expressionconstructs to patients with 3p21.3-linked cancers will be a veryefficient method for delivering a therapeutically effective gene tocounteract the clinical disease. Similarly, the chemo- or radiotherapymay be directed to a particular, affected region of the subjects body.Alternatively, systemic delivery of expression construct and/or theagent may be appropriate in certain circumstances, for example, whereextensive metastasis has occurred.

In addition to combining Fus1, 101F6, Gene 21, Gene 26, Beta*, Luca1,Luca2, PL6, 123F2, or SEM A3-targeted therapies with chemo- andradiotherapies, it also is contemplated that combination with other genetherapies will be advantageous. For example, targeting of Fus1, 101F6,Gene 21, Gene 26, Beta*, Luca1, Luca2, PL6, 123F2, or SEM A3 and p53 orp16 mutations at the same time may produce an improved anti-cancertreatment. Any other tumor-related gene conceivably can be targeted inthis manner, for example, p21, Rb, APC, DCC, NF-1, NF-2, BCRA2, p16,FHIT, WT-1, MEN-I, MEN-II, BRCA1, VHL, FCC, MCC, ras, myc, neu, raf,erb, src, fms, jun, trk, ret, gsp, hst, bcl and abl.

It also should be pointed out that any of the foregoing therapies mayprove useful by themselves in treating a Fus1, 101F6, Gene 21, Gene 26,Beta*, Luca1, Luca2, PL6, 123F2, or SEM A3-related disorder. In thisregard, reference to chemotherapeutics and non-Fus1, 101F6, Gene 21,Gene 26, Beta*, Luca1, Luca2, PL6, 123F2, or SEM A3 gene therapy incombination should also be read as a contemplation that these approachesmay be employed separately.

4. Formulations and Routes for Administration to Patients

Where clinical applications are contemplated, it will be necessary toprepare pharmaceutical compositions—expression vectors, virus stocks,proteins, antibodies and drugs—in a form appropriate for the intendedapplication. Generally, this will entail preparing compositions that areessentially free of pyrogens, as well as other impurities that could beharmful to humans or animals.

One will generally desire to employ appropriate salts and buffers torender delivery vectors stable and allow for uptake by target cells.Buffers also will be employed when recombinant cells are introduced intoa patient. Aqueous compositions of the present invention comprise aneffective amount of the vector to cells, dissolved or dispersed in apharmaceutically acceptable carrier or aqueous medium. Such compositionsalso are referred to as inocula. The phrase “pharmaceutically orpharmacologically acceptable” refer to molecular entities andcompositions that do not produce adverse, allergic, or other untowardreactions when administered to an animal or a human. As used herein,“pharmaceutically acceptable carrier” includes any and all solvents,dispersion media, coatings, antibacterial and antifungal agents,isotonic and absorption delaying agents and the like. The use of suchmedia and agents for pharmaceutically active substances is well know inthe art. Except insofar as any conventional media or agent isincompatible with the vectors or cells of the present invention, its usein therapeutic compositions is contemplated. Supplementary activeingredients also can be incorporated into the compositions.

The active compositions of the present invention may include classicpharmaceutical preparations. Administration of these compositionsaccording to the present invention will be via any common route so longas the target tissue is available via that route. This includes oral,nasal, buccal, rectal, vaginal or topical. Alternatively, administrationmay be by orthotopic, intradermal, subcutaneous, intramuscular,intraperitoneal or intravenous injection. Such compositions wouldnormally be administered as pharmaceutically acceptable compositions.Upon formulation, solutions will be administered in a manner compatiblewith the dosage formulation and in such amount as is therapeuticallyeffective.

EXAMPLES

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skilled the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the concept, spirit andscope of the invention. More specifically, it will be apparent thatcertain agents which are both chemically and physiologically related maybe substituted for the agents described herein while the same or similarresults would be achieved. All such similar substitutes andmodifications apparent to those skilled in the art are deemed to bewithin the spirit, scope and concept of the invention as defined by theappended claims.

Example 1 Identification of 3p Genes in 3p21.3 Critical Region andIsolation of cDNA of 3p Genes

The 3p tumor suppressor region was identified by allelotyping designedto search for areas of LOH in matched tumor/normal tissue pairs, andexamine uncommon examples of homozygous deletions (FIG. 4). The mostfrequently involved region showing allele loss in lung cancer was mappedto the 3p21.3 region. Furthermore, multiple overlapping homozygousdeletions have been found in the 3p21.3 chromosome region in SCLC linesH740 and H1450, which narrowed down the search for the tumor suppressorgenes flanking about 750 kb in 3p21.3 region. Nine genes, Fus1, 101F6,NPRL2 (Gene 21), CACNA2D2 (Gene 26), HYAL1 (Luca 1), HYAL2 (Luca 2),PL6, 123F2, and Beta*, were either disrupted or immediately flanking a˜35 kb homozygous deletion found in the 3p21.3 region. SEM A3 is alsopresent in the 3p21.3 region. The cDNAs of these genes were isolated andcloned, and mutations in these genes were determined in various tumorand tumor cell lines by single strand conformation polymorphism (SSCP)and DNA sequencing analysis (Table 4). Some of the cDNA clones showed˜50% amino acid homologies to known genes in the GeneBank, somedemonstrated complete DNA sequence homology to random sequence taggedsites in the GeneBank, and one gene, Beta*, was previously unknown(Table 4).

TABLE 4 Genes Identified in the 125 kb 3p21.3 Critical Region and Statusof Their cDNA Sequencing and Mutation Analysis GenBAnk CDNA Sequence(bp) Mutation Analysis Gene* Number (aa) (Numbers) Mutations** CACNA2D2(Gene 26) AF040709 5,482 bp (1,205 aa) Yes (60) none PL6 U09584 1,860 bp(351 aa) Yes (38) none 101F6 AF040709 1,117 bp (222 aa) Yes (38) noneNPRL2 (Gene 21) AF040707, 1,696 bp (203 aa) Yes (38) 1 stop AF040708Beta* (BLU) none 1,739 bp (440 aa) Yes (61) 3 missense 123F2 (RASSF1)AF040703 1,502 bp (431 aa) Yes (37) none FUS-1 AF055479 1,696 bp (161aa) Yes (79) 2 stop HYAL2 (LUCA-2) U09577 1,783 bp (473 aa) Yes (40)none HYAL1 (LUCA-1) U03056 2,565 bp (435 aa) Yes (40) 2 missense *Thepredicted amino acid sequence homologies to other known genes include:CACNA2D2 (Gene 26), voltage gated Ca²⁺ channel alpha 2 delta regulatorysubunit; NPRL2 (Gene 21), nitrogen permease regulator; 123F2,Maxp1/Norel homologue of a Ras binding protein; HYAL2 (LUCA-2) and HYAL1(LUCA-1), a family of hyaluronidases. **Only mutations altering the amioacid sequence are shown. In addition, polymorphisms found in more thanone tumor and that did not alter the amino acid sequence are not given.

Example 2 Construction of Recombinant Adenoviral Vectors of 3p Genes

Adenoviral vectors have been widely used for gene delivery in vitro, inanimal models, preclinical research, and human clinical gene therapytrials. The high efficiency of transduction and high-level expression oftransgenes mediated by adenovirus vectors in various cell types arereasons why the recombinant adenoviral vectors expressing the 3p genes(Ad-3ps) are effective tools for introduction of the functionalwild-type 3p genes into tumors or tumor cell lines with abnormalities of3p or 3p genes.

Recombinant adenoviral vectors of 3p21 genes, including Gene21, Fus1,101F6, Gene 26, 123F2S, Luca1, and Beta*, have been constructed usingthe inventors' recently developed ligation-mediated plasmid-adenovirusvector construction system, pAd-RAP and pAd-RAP-Shuttle. The inventorshave successfully and rapidly constructed recombinant adenoviral vectorsfor all ten genes in the 3p21.3 region and many other recombinantvectors using this system. Recombinant Ad-3ps can efficiently deliver 3pgenes into and express them in various cell types in vitro by directlyinfecting target cells and in vivo by intravenous or local injection ofvectors. The relative genomic locations of the tumor suppressor 3p21.3genes in chromosome 3p and the structure of the recombinant adenoviralvectors of 3p genes are schematically demonstrated (FIG. 5).

The inventors have developed a novel ligation-mediatedplasmid-adenovirus vector construction system, named pAd-RAP andpAd-RAP-Shuttle. This system can be used to rapidly constructrecombinant adenovirus-containing plasmids in bacterial Escherichiacoli, and then successfully produce homogeneous adenovirus in mammalianhost 293 cells (FIG. 1). In this system, the transgene (X) is firstplaced in a plasmid shuttle vector, pAd-RAP-Shuttle, containing theadenoviral inverted repeated terminal (IRT) sequence, an expressioncassette of a cytomegalovirus (CMV) promoter and bovine growth hormone(BGH) poly (A) signal sequence, and two unique restriction sites BstBIand ClaI at the 5′ and 3′ ends of the IRT-CMV-multiple cloning sites-BGHsequence, respectively. The BstBI/ClaI-released DNA fragment containingIRT-CMV-X-BGH is then inserted into an adenoviral plasmid vector,pAd-RAP, which contains a complete E1 and E3-deleted adenovirus type 5genome and three unique restriction sites (Pact, BstBI, and ClaI), by invitro ligation using BstBI and ClaI sites. After transformation intoEscherichia coli, 90% of the transformants have the correct insert.Finally, PacI/BstBI digestion of the resulting plasmid allows release ofthe entire adenovirus genome-containing the 3p gene. The recombinantAd-X DNA is then transfected into 293 cells, resulting in a homogeneouspopulation of recombinant Ad-X. Other promoters, poly A sequences, andrestriction sites can be used. This system can be used to rapidlyconstruct a recombinant adenovirus within 2-4 weeks. By comparison, theconventional methods, such as that using homologous recombination inmammalian cells, will usually take 3-12 months.

In case of failure to produce a specific recombinant Adenovirus due tothe possible cytotoxicity of the transgenes in the host 293 cells, asimilar system named pAd-RAP-TetR-Off and pAd-RAP-TRE-CMV-Shuttle, asdemonstrated in FIG. 2, with tetracycline regulatory elements (TRE) thatcan turn off transgene expression in the presence of tetracycline hasbeen developed and can be used for the production of such vectors.

The Ad-CMV-GFP (Ad-GFP) and Ad-CMV-LacZ vectors were used to monitortransduction efficiency by the viral vectors and as nonspecifictransgene expression controls. Ad-E1-(Ad-EV), an empty E1-vector, isused as a negative control. Viral titers were determined by both opticaldensity measurement and plaque assay. Potential contamination of theviral preparation by the wild-type virus was monitored by polymerasechain reaction (PCR) analysis. Sequences of 3p genes in the viralvectors were confirmed by automated DNA sequencing. The resulting Ad-3psare named, Ad-101F6, Ad-Gene26, Ad-Gene21, Ad-Fus1, Ad-PL6, Ad-Luca1,Ad-Luca2, Ad-123F2S, Ad-Beta*, and Ad-SEM A3, respectively.

Example 3 Preparation of PAD3ps

The preparation of protamine-adenovirus (PAD) complexes and enhancementof transduction efficiency by PADs in vitro and in vivo have beenreported^(47,48). The protamine-adenovirus complexes were prepared bysimply mixing 1×10¹⁰ viral particles with 50 μg of protamine sulfate (10mg/ml). The complexes were incubated for 10 min at room temperature, andthen the complexed adenovirus were diluted in an appropriate volume ofPBS for designated in vitro or in vivo experiments.

Example 4 Preparations of LDC3ps and LPD3ps

The liposome (DOTAP:Cholesterol) (LDC), plasmid DNA, and LDC-3p DNAcomplexes (LDC3ps) were prepared as described by Templeten et al.⁴⁹.LDC3ps were formulated as 80 nmol liposome:50 μg DNA in 5% Dextral water(D5W) at a total volume of 100 μl for intravenous injection to onemouse. Liposome (DOTAP:Cholesterol):Protamine:DNA (LPD) were preparedbased on the method of Hung⁵⁰.

Example 5 Effects of Overexpression of 3p Genes on Tumor Cell Growth

To study biological function of new tumor suppressor genes, experimentsare conventionally performed in tumor cell lines either transiently orstably transfected by wild type gene-expressing plasmids. The Ad-3pvectors can offer several advantages over plasmids for 3p gene deliveryin vitro and in vivo: 1) high efficiency (>80%) of transduction and highlevel of 3p gene expression can be easily achieved in a wide spectrum ofcell types by simply adjusting the multiplicity of infection (M01) ofviral particles to target cells, consequently, the Ad-3ps can be used toevaluate effects of 3p genes as a individual or as a whole region 2)Ad-3ps-transduction can be directly applied to tumor cells to studytheir effect on tumorigenicity in animals without selection of stablytransduced colonies, by which problems associated with colony selectionprocess and unknown effectors or factors generated in resulting cellcolonies can be avoided; and 3) Ad-3ps can be directly used to evaluatethe role of 3p genes as a tumor suppressor gene region in vivo by eitherintravenous or intratumoral injection of animals with the individual orcombined Ad-3p vectors.

The biological function of these newly isolated 3p genes ischaracterized in this invention by liposome- and recombinant adenoviralvector-mediated gene transfer both in vitro and in vivo. Human lungcancer cell lines (H1299, H358, H460, and A549), with varied status ofchromosome 3p or individual 3p genes and a normal human bronchialepithelial cell (HBEC) line were used to evaluate the effects of 3pgenes on cell growth arrest, proliferation, apoptosis, and cell cyclekinetics in vitro and on growth of the primary and metastatic tumors inanimal models.

To test the hypothesis that the 3p genes function as tumor suppressorsin vitro, the inventors performed a series of experiments to study theeffects of overexpression of the 3p genes on cell proliferation invarious human non-small cell lung cancer cells and a normal humanbronchial epithelial cell line (HBEC) varying in status of 3pchromosomal structure or genes and gene products (Table 5) by liposome-or adenoviral vector-mediated 3p gene transfer. One of these lines isH1299, a NSCLC cell line that contains an internal homozygous deletionof p53, and has no normal copy of chromosome 3 with LOH of 3p allelesand has very high levels of telomerase expression and activity. A549, isa lung carcinoma cell line that contains wild-type p53 with abnormal 3palleles; H358 is a lung cancer cell line that contains wild-type p53with two 3p alleles; and H460 is, a lung cancer cell line that containswild-type p53 with loss of noe allele of the 3p21.3 region (Table 5).Normal HBECs or fibroblast cells (Clonetics Inc., Walkersville, Md.)were also used to evaluate the general toxicity of the 3p genes andAd-3ps. The 293 cell line was used in the construction, amplification,and titration of adenoviral vectors. The cells were maintained inDulbecco's Modified Eagle Medium (DMEM) containing 4.5 g/l of glucosewith 10% FBS.

TABLE 5 Status of 3p Genes in Human Lung Cancer Cell Lines and NormalHBEC* Cell Line Origin 3p Genes P53 hTERT H1299 Lung, large LOH DeletionHigh Activity A549 NSCL LOH WT Active H460 Lung, Large LOH WT ActiveH358 NSCL WT Deletion Active HBEC Bronchial Epithelia Normal Normalundetected *Abbreviation: HBEC, human bronchial epithelia cell, NSCL,non-small cell lung cancer, LOH, loss of heterozygosity, WT, wild type.

The Ad-3p vectors, protamine-Ad-3p complexes (PAD3ps) or liposome(DOTAP)-3p plasmid DNA complexes (LPD3ps) developed in this inventioncan be used to deliver 3p genes efficiently to the tumor cells in vitroby direct transduction and to the primary and distant lung or othermetastatic tumor sites in vivo by systemic administration. Thespontaneous or experimental pulmonary metastasis models of human lungcancers H1299 and A549, as well as other cancers, can be used to studythe effects of 3p genes on tumor progression and metastasis by systemictreatment of lung metastatic tumors in mice through intravenousinjection of either PAD3p or LPD3p complexes.

In experiments with liposome-mediated 3p gene transfer in H1299 cells,six genes out of the nine, Fus1, 101F6, Luca1, 123F2S, Beta*, and Gene21, demonstrated varied degrees (20-65%) of cell growth inhibition inH1299 transfectants after 48 hr of transfection, compared tountransfected and empty CMV vector-transfected controls. Three othergenes Gene 26, PL6, and Luca 2 showed no significant effects on H1299cell growth under the same experimental Condition (Table 6). Theobserved inhibitory effect of Fus1, Beta*, 123F2S, and Gene 21 on H1299cell growth were comparable to that of highly cytotoxic gene Bak underthe same experiment conditions (Table 6). The three other genes Gene 26,PL6, and Luca 2 showed no significant effects on H1299 cell growth underthe same experimental conditions. Varied degrees (10-40%) of inductionof apoptosis and altered cell cycle kinetics (changes of cellpopulations at G0, G1 and S phases) were observed in H1299 cellstransfected with plasmids containing genes Fus1, 101F6, 123F2S, Luca,and Beta* by FACS analysis with TUNEL reaction and PI staining.

TABLE 6 Effects of DOTAP-mediated 3p Gene Expression on Growth of H1299Cells (48 h) Data from MDACC Data from UTSMC (Transfection) (ColonyFormation)^(†) (% Cell (% Cell Viability ± STDEV) Viability ± STDEV) PBS100 ND CMV-EV 85 ± 9.5 100 GFP 84 ± 7.2 ND Bak 54 ± 5.1 ND 101F6 76 ±3.5 52 ± 10.0 Fus1 78 ± 2.1 49 ± 14.0 Gene 21 45 ± 6.5 83.7 ± 17.7  Gene26  88 ± 12.5 40 ± 0.00 Luca 1 81 ± 2.8 66 ± 27.2 Luca 2 100 ± 9.8  80 ±27.6 PL6 100 ± 13.6 95 ± 53.8 123F2S 67 ± 3.8 58 ± 0.0  Beta* 35 ± 2.351 ± 8.5  ^(†)Data from colony formation assay in 3p gene-expressingplasmid DNA-tranfected cells, relative to that of emptyplasmid-transfected cells. ND, undetermined; STDEV, standard deviationfrom the mean of the repeated experiments.

Effects of 3p genes on tumor cell growth were further characterized byrecombinant-adenoviral vector-mediated 3p gene transfer in various lungcancer cell lines and a normal HBEC line. To test the specificity of theobserved inhibitory effects of 101F6, Fus1, and Gene21 overexpression ontumor cell proliferation and the potential cytotoxicity of theoverexpressed 3p genes, the inventors analyzed the effect of these 3pgenes on cell proliferation in Ad-3p-transduced wild type 3p-containingH358 cells and normal HBEC cells (FIG. 6). Cells in each line weretransduced in vitro by Ad-101F6, Ad-fus1 and Ad-Gene21 vectorsadministered at various multiplicity of infections (MOIs) in viralparticles/cell (vp/c); cells treated with PBS were used as mock, emptyvector Ad-EV as negative, Ad-LacZ as nonspecific, and Ad-p53 as positivecontrols, respectively. A less than 10% loss of cell viability inAd-3p-transduced HBEC and a less than 20% loss in H358 cells at variousMOIs, were observed when compared with that in untransduced controlcells. Similar losses were also observed in Ad-EV- and Ad-LacZtransduced cells and slightly higher loses in Ad-p53-transduced cellsthroughout the posttransduction time course, suggesting that nogeneralized cytotoxicity was associated with overexpression of these 3pgenes. The transduction efficiency was determined by examining theGFP-expressing cells in the Ad-GFP transduced cell population under afluorescence microscope.

The transduction efficiency of the adenoviral vectors was greater than80% at the highest MOI applied for each cell line. Cell proliferationwas analyzed by determining the viability of cells at 1, 3 and 5 daysposttransduction, respectively. Cell viability was significantly reducedin Ad-101F6, Ad-Fus1, and Ad-Gene21 transduced A549 and H460 cells whichexhibit LOH in 3p region but contain wild-type p53 and H1299 cells whichcontains homozygous deletions of 3p region and p53 (FIG. 6). In allcases, the viability of transduced cells was compared with that ofuntransduced (PBS-treated) control cells (whose viability was set at100%).

The overexpression of 3p genes in these Ad-3p tranfectants was verifiedby a quantitative Real Time RT-PCR, and known concentrations of humantotal RNA, primers, and TaqMan probes for β-actin DNA were used asstandards and as a internal control (FIG. 7). TaqMan probes and primersof 3p genes were designed using a Primerexpress software (Perkin ElmerApplied Biosystems, Foster City, Calif.). Human genomic DNA or totalRNAs were used as template standards and human β-actin orglyceraldehyde-3-phosphate dehydrogenase (GAPDH) TaqMan probes andprimers as controls. Total RNA was isolated from Ad-3p transduced tumorcells or tumor specimen using TRIZOL. Real time RT-PCR andquantification of RT-PCR products were performed and analyzed using aTaqMan Gold RT-PCR Kit, an ABI Prism 7700 Sequence Detection System andequipped software. These results show that overexpression of these 3pgenes can inhibit tumor cell growth in vitro.

Example 6 Effects of 3p Genes on Tumor Cell Growth and Proliferation

To test whether the growth properties of various lung cancer cells withabnormalities of 3p or 3p genes could be altered by the introduction ofwild-type 3p genes, cell viability in Ad-3p-transduced tumor cells atvaried MOIs at designated posttransduction time intervals are assayed byXTT staining as described previously,⁴⁴ and the untransduced and Ad-EV-,Ad-GFP-, or Ad-LacZ-transduced cells were used as controls. Eachexperiment was repeated at least three times, with each treatment givenin duplicate or triplicate. Proliferation of the Ad-3p-transduced cellswere analyzed by an immunofluorescence-enzyme-linked immunosorbent assayfor incorporation of bromodeoxyuridine (BrdU) into cellular.Ad-3p-transduced normal HBECs were used to evaluate the possible generaltoxicity of the 3p genes and Ad-3ps in vitro. Transcription andexpression of 3p genes in Ad-3p-transduced cells were examined byreverse transcriptase-polymerase chain reaction or Northern- andWestern-blot analysis with anti-3p protein polyclonal antibodies.

Example 7 Western Blot Analysis of Expression of 3p Genes inAd-3p-Transduced Cells

Expression of 3p genes in Ad-3p-transduced cells was analysed by Westernblot, using polyclonal antibodies against polypeptides derived frompredicted 3p amino acid sequences or monoclonal antibodies against c-mycof FLAG tags in 3p fusion proteins. Cells grown in 60 mm-dishes(1-5×10⁶/well) were treated with Ad-3ps, and PBS alone was used as acontrol. Proteins were separated by SDS-PAGE. Each lane was loaded withabout 60 μg cell lysate protein and electrophoresed at 100 V for 1-2 h.Proteins were then transferred from gels to Hybond-ECL™ membranes.Membranes were blocked in blocking solution (3% dry milk, 0.1% Tween 20in PBS) for 1 h at room temperature. Membranes were then incubated with1:1000 dilution of rabbit anti-human 3p peptides or anti-myc or FLAGmonocolonal antibodies, and 1:1000 dilution of mouse anti-β-actinmonocolonal antibodies. Immunocomplexes were detected with secondaryHRP-labeled rabit anti-mouse IgG or goat anti-rabit IgG antibodies usingan ECL™ kit (Amersham International).

Example 8 Induction of Apoptosis by 3p Genes in Ad-3p-Transduced TumorCells

The ability of exogenous 3p genes to induce apoptosis and their impacton cell-cycle processes in the Ad-3p-transduced H1299, A549, H460, H358,and HBEC cells were analyzed by FACS using the terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL) reaction (FIG. 8).Induction of apoptosis was detected in Ad-3p-transduced H1299 (FIG. 8A),A549 (FIG. 8B), and H460 (FIG. 8C) cells, but not in H358 (FIG. 8D) andHBEC (FIG. 8E) cells. More than 15-20%, 40-65%, and 75% of cells wereapoptotic at day 5 after transduction with Ad-101F6, Ad-Fus1, andAd-Gene21 in the transduced H1299, A549, and H460 cells, respectively,whereas only about 7% and 10% of cells treated with PBS alone andtransduced with Ad-EV vector, respectively, were TUNEL-positive at thesame time periods. The level of induction of apoptosis in theAd-3p-transduced cells increased with time posttransduction andcorrelated with the viability of cells (FIG. 6). The inhibition of tumorcell proliferation by 3p genes are mediated directly or indirectlythrough induction of apoptosis.

Example 9 Induction of Apoptosis and Alteration of Cell Cycle KineticsAd-3p-Transduced Cells

Inhibition of tumor cell growth and proliferation by tumor suppressorgenes is usually characterized by induction of apoptosis and alterationof cell cycle processes. Thus, 3p gene-induced apoptosis and cell cyclekinetics were analyzed by flow cytometry using the terminal deoxytransferase deoxyuridine triphosphate (dUTP) nick-end labelling (TUNEL)reaction with fluorescein isothiocyanate-labeled dUTP (Roche MolecularBiochemicals) and propidium iodide staining, respectively. In brief,cells (1×10⁶/well) are seeded on six-well plates and transduced withAd-3p constructs; untreated and Ad-EV-, Ad-GFP-, or Ad-LacZ-transducedcells were used as controls. Cells were harvested at designatedposttransduction times and then analyzed for DNA fragmentation andapoptosis by TUNEL reaction and for DNA content and cell cycle status bypropidium iodide staining using flow cytometry, respectively, asdescribed previously. The cell-cycle profiles in the Ad-101F6, Ad-Fus1,and Ad-Gene21-transduced cells appeared to be significantly affected byoverexpression of these genes at later G2 and S phases stages comparedto those in the untransduced and Ad-EV-transduced controls at 3 daysposttransduction (FIG. 9).

Example 10 Suppression of Tumor Growth by Overexpression of 3p Genes InVivo

The tumor suppressor function of 3p genes, 101F6, Fus1, and Gene21 wereevaluated in vivo by direct intratumoral injection of these Ad-3pvectors into the A549 subcutaneous tumors in nude mice (FIG. 10). Thegrowth of tumors was recorded from first injection until 20 days afterlast injection. All of the tumors in the mice treated with Ad-101F6,Ad-Fus1, and Ad-Gene21 showed significantly suppressed growth comparedwith tumors.

Example 11 Effects of 3p Gene Expression on Tumorigenicity and TumorGrowth In Vivo

For the tumorigenicity study, H1299 or A549 cells were transduced invitro with Ad-3p at an appropriate MOI with phosphate-buffered saline(PBS) alone as a mock control, Ad-EV as a negative control, and Ad-LacZas a nonspecific control. The transduced cells were harvested at 24 hand 48 h posttransduction, respectively. The viability of the cells wasdetermined by trypan blue exclusion staining. Viable cells (1×10⁷) werethen injected subcutaneously into the right flank of 6- to 8-week-oldfemale nude mice. Tumor formation in mice was observed two or threetimes weekly for up to 3 months. Tumor dimensions were measured every 2or 3 days.

To study the effect of 3p genes on tumor growth, H1299 or A549 cellswere used to establish subcutaneous tumors in nude mice. Briefly, 1×10⁷cells were injected into the right flank of 6- to 8-week-old female nudemice. When the tumors reached 5 to 10 mm in diameter (at about 2 weekspostinjection), the animals were intratumorally injected with Ad-3p andcontrol vectors, respectively, 4 to 5 times within 10 to 12 days for ata total dose of 3 to 5×10¹⁰ pfu per tumor. Tumor size was measured andcalculated as described above. At the end of the experiment, the animalswere killed and the tumors were excised and processed for pathologicaland immunohistochemical analysis.

Example 12 Inhibition of Lung Metastatic Tumor Growth byProtamine-Adenovirus Complex-Mediated 3p Gene Transfer In Vivo

The inventors have developed a novel Protamine/Adenovirus complex forenhancement of the efficiency of adenovirus-mediated gene transfer invitro and for systemic delivery of recombinant adenovirus to lung andother organs in vivo by intravenous injection of the complex. TheProtemine-Ad-3p complexes (PAd3ps) were used to study the effects ofoverexpression of 3p genes on pulmonary metastatic tumor growth in A549experimental lung metastasis model in nude mice (FIG. 11). Themetastatic tumor growth was significantly inhibited in PAd-101F6,PAd-Fus1, and PAd-Gene21-treated mice, compared with those in controlgroups. These data are consistent with results obtained fromAd-3p-treated subcutaneous tumors. Therefore, the 3p genes play a rolein suppression of tumor growth and inhibition of tumor progression invivo.

Example 13 Effect of 3p genes on metastatic tumor growth by LPD3p- orPAD3p-Mediated 3p Genes Transfer In Vivo

The experimental lung metastasis models of H1299 and A549 cells wereused to study the effects of 3p genes on tumor progression andmetastasis by systematic treatment of lung metastatic tumors throughintravenous injection of either PAD3p or LPD3p complexes. A549 cells(1-2×10⁶) in 200 μl PBS were intra venially inoculated with nude miceand H1299 cells (1-2×10⁶) with SCID mice, respectively. Experimentalmetastatic tumor colonies were formed 7-10 days post-inoculation. PAD3psand control complexes were administered to animals by intravenousinjection every other two days for 3 times each at a dose of 2-5×10¹⁰viral particles/200-500 μg protamine, in a total volume of 200 μl peranimal. Alternatively, LPD3ps were applied by intravenous injectionevery day for 6 times each at a dose of 120 nmol liposome:6 μgprotamine:50 μg DNA, in a total volume of 200 μl per animal. Animalswere killed two weeks post last injection. Lung metastasis tumors werestained with Indian ink⁵¹, tumor colonies on the surfaces of lung werecounted under an anatomic microscope, and then the lung tissue weresectioned for further pathologic and immunohistochemical analysis.

Example 14 Analysis of Telomerase Activity and Cellular Immortality

Activation of the enzyme telomerase, which has been associated withcellular immortality, may constitute a key step in the development ofhuman cancer. Because of the nearly universal deregulated expression oftelomerase in lung cancer cells and the evidence for involvement of 3pgenes in the telomerase repression regulatory pathway, it will beimportant to study whether the alteration of tumor cell growth andproliferation implied by the introduction of wild-type 3p21.3 genes isassociated with repressed telomerase activity in Ad-3p transductants. Toassay telomerase activity, untransduced and Ad-3p- and controlvector-transduced cells (10⁵) are harvested and prepared as describedpreviously.³¹ The cell extract equivalent to approximately 10³, 10², or10¹ cells is used for each telomerase assay. A standard telomeric repeatamplification protocol procedure, which is capable of detectingtelomerase activity in as few as 10 to 100 lung cancer cells, isperformed with modifications as described.^(32,45,46)

Example 15 123F2 (RASSF1A) in Lung and Breast Cancers and MalignantPhenotype Suppression³³⁹ I. Characterization of the 123F2 (RASSF1) Gene

To determine if the 123F2 (RASSF1A) gene was mutated in lung and breastcancers, the inventors performed extensive mutational analysis of theRASSF1A isoform with the use of single-strand conformation polymorphismassays on genomic DNA. The inventors had previously found no RASSF1Cmutations in 77 lung cancer cell line samples³⁴⁶. By use of the RASSFIAsequence as a reference, the inventors found several polymorphisms,including the following: codon 21 (AAG to CAG), Lys to Gln; codon 28(CGT to CGA), no amino acid change; codon 49 (GGC to GGT), no amino acidchange; codon 53 (CGC to TGC), Arg to Cys; codon 129 (GAC to GAG), Aspto Glu; codon 133 (GCT to TCT), Ala to Ser; and codon 325 (TAT to TGT),Tyr to Cys. The 123F2 (RASSF1) gene is shown in FIG. 12.

II. Expression of RASSFIA and RASSF1C in Lung and Breast Cancer CellLines

123F2 (RASSF1) is located within a region frequently affected by alleleloss during growth of lung, breast, head and neck, kidney, and cervicaltumors³⁴¹⁻³⁴⁵. The inventors investigated whether 123F2 (RASSF1A) andRASSF1C are expressed in lung and breast cancer cell lines. Theinventors used isoform-specific RT-PCR to examine the expression of123F2 (RASSF1A) and RASSF1C in lung and breast tumor cell lines and innormal lung and breast epithelial cultures (FIG. 13).

Isoform-specific RT-PCR assays were used for analysis of RASSF1A andRASSF1C expression. Primers for RASSFIC were Nox3(5′-CTGCAGCCAAGAGGACTCGG-3′) and R182 and for RASSF1A were either PKCDFor NF (5′-TGCAAGTTCACCTGCCAC-3′) and R182 (FIG. 12. C). Total RNA wasisolated from previously described lung and breast cancer cell linesgrown in RPMI-1640 medium supplemented with 5% fetal bovine serum(complete medium) by Trizol extraction. Four micrograms of total RNA wasreverse transcribed by use of GIBCO-BRL Superscript First Strand cDNAKit. All cDNA preparations were tested for the ability to amplify anontranscribed genomic sequence immediately upstream of the first exonof the RASSF1A transcript. Any cDNAs that produced a product from thissequence were discarded because they were contaminated with genomic DNA.

The inventors also assessed the expression of RASSF1A after exposure to5-aza-2′-deoxycytidine, a drug that inhibits DNA methylation. Theinventors exposed subconfluent cultures of the RASSF1A-nonexpressingNSCLC line NCI-H157 to 0.5 p.M 5-aza-2′-deoxycytidine for 48 hours,after which the inventors isolated total RNA and performed RT-PCR forRASSF1A, RASSF1C, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH).RT-PCR of GAPDH transcripts was performed with the use of forward primerGAPDH-C (5′-CATGACAACTTTGGTATCGTG-3′) and reverse primer GAPDH-D(5′-GTGTCGCTGTTGAAGTCAGA-3′). RTPCR products were separated by agarosegel electrophoresis and visualized after staining with ethidium bromide.

123F2 (RASSF1A) was expressed in normal lung epithelial cultures (NHBEand SAB cultures), in a normal breast epithelial culture (FIG. 13, C),but not in 32 (100%) of 32 SCLC lines, in 17 (65%) of 26 NSCLC celllines, and in 15 (60%) of 25 (60%) breast cancer cell lines.Representative data are shown in FIG. 13. By contrast, RASSF1C wasexpressed in nearly all of the lung and breast cancer cell lines tested,with the exceptions of several lung and breast cancer lines with knownhomozygous deletions that include the 123F2 (RASSF1) locus. In resectedlung adenocarcinomas, 123F2 (RASSF1A) was expressed in only two of fivecancers, while RASSF1C was expressed in all cancers (FIG. 13, C).

During RT-PCR analysis for 123F2 (RASSF1A), the inventors frequentlynoted two closely spaced bands in RASSF1A-expressing tumors and in NHBEcultures (FIG. 13). The inventors sequenced these RT-PCR products andfound that the larger band corresponded to 123F2 (RASSF1A), while thesmaller product represented a different transcript, RASSF1F (GenBankAccession #AF286217). This transcript skips exon 1C to produce an mRNAencoding a predicted truncated peptide of 92 amino acids ending withinthe DAG-binding domain (FIG. 12. D). In nearly all of the samples,RASSF1F is expressed when 123F2 (RASSF1A) is expressed. However, in somebreast cancers and normal breast epithelial cultures (FIG. 13), 123F2(RASSF1A) is expressed without RASSF1F expression.

III. Methylation Status of the 123F2 (RASSF1A) Promoter Region

Aberrant promoter methylation in tumors has been found to lead to theloss of gene expression of several tumor suppressor genes in humancancers'. To assess whether the loss of 123F2 (RASSF1A) expression inlung cancer was the result of promoter hypermethylation, the inventorsdetermined the CpG methylation status in the 5′ region of 123F2(RASSF1A) (from −800 to +600 bp of the predicted 123F2 (RASSF1A)transcript start site) by sequencing sodium bisulfite-modified DNA fromeight lung cancer cell lines.

The methylation status of the presumed RASSF1A and RASSF1C promoterregions was determined by methylation-specific PCR. Genomic DNAs fromlung cancer cell lines not expressing RASSF1A (NCI lines H1299, H1184,H1304, H841, H2108, and H128) or expressing RASSF1A (H1792 and H2009)were modified by sodium bisulfite treatment^(352, 353). Bisulfitetreatment converts cytosine bases to uracil bases but has no effect onmethylcytosine bases. PCR amplification followed by sequencing of thePCR fragments identifies specific CpG dinucleotides in the promoterregion that are modified by methylation^(352, 354, 355). PCR primerswere designed to amplify genomic sequences in the presumed promoterregions of RASSF1A (cosmid Luca12; GenBank Accession #AC002481nucleotides 17730-18370) and RASSF1C (GenBank Accession #AC002481nucleotides 21022-21152 and 21194-21332). The resulting PCR fragmentswere sequenced by automated fluorescence-based DNA sequencing todetermine the methylation status.

The data on CpG methylation in RASSF1A-nonexpressing lung cancer celllines were used to design methylation-specific PCR³⁵² primers for theRASSF1A 5′ promoter region: The primers to detect the methylated formwere 5′-GGGTTTTGCGAGAGCGCG-3′ (forward) and 5′-GCTAACAAACGCGAACCG-3′(reverse), and the primers to detect the unmethylated form were5′-GGTTTTGTGAGAGTGTGTTTAG-3′ (forward) and 5′-CACTAACAAACACAAACCAAAC-3′(reverse). Each primer set generated a 169-base-pair (bp) product.Methylation-specific PCR cycling conditions consisted of one incubationof 15 minutes at 95 'C, followed by 40 cycles of a 30-seconddenaturation at 94° C., 50 seconds at an annealing temperature (64° C.for methylation-specific and 59° C. for unmethylated-specific primers),a 30-second extension at 72° C., and a final extension at 72° C. for 10minutes. PCR products were separated in 2% agarose gels. Lymphocyte DNA,methylated in vitro by CpG (SssI) methylase (New England Biolabs, Inc.,Beverly, Mass.) following the manufacturer's directions, was used as apositive control. A water blank was used as a negative control.

All of the six lung cancer cell lines not expressing 123F2 (RASSF1A)exhibited methylation of almost all CpG dinucleotide sites in theputative promoter region. The two lung cancer cell lines that didexpress 123F2 (RASSF1A either were not methylated at these CpG sites orshowed limited methylation. By contrast, no methylation was found in CpGsites in the presumed RASSF1C promoter region of these eight cell lines.

To confirm that promoter hypermethylation contributes to the lack ofexpression of 123F2 (RASSF1A) in the lung cancer cell lines, theinventors assessed the effect of 5-aza-2′-deoxycytidine, a drug thatinhibits DNA methylase, on 123F2 (RASSF1A) expression. The inventorsexposed the RASSF1A-nonexpressing NSCLC line NCI-H157 to5-aza-2′-deoxycytidine and found re-expression of 123F2 (RASSF1A) bythis cell line but little or no change in the expression of thehousekeeping gene GAPDH or in the expression of RASSF1C (FIG. 14).

IV. Methylation-Specific PCR Analysis of the Promoter Region of 123F2(RASSF1A) in Lung and Breast Cancers

To determine the methylation status of the promoter region of RASSE1A inprimary lung and breast cancers, the inventors used methylation-specificPCR analysis. Genomic DNA from a large number of primary resectedNSCLCs, paired lung tissues resected from the same patients but notinvolved with the cancer, primary resected breast cancers, and a largepanel of lung and breast cancer cell lines were treated with sodiumbisulfite and tested for the presence of methylated and unmethylated CpGdinucleotides in the promoter region of 123F2 (RASSF1A) (FIG. 15). Allof the primary resected NSCLCs and non-tumor-paired samples containedunmethylated promoter sequences, which were expected because theseresected tumors were not microdissected and were contaminated withstromal cells. However, 32 (30%) of 107 primary NSCLCs, 47 (100%) of 47SCLC lines, and 19 (49%) of 39 primary breast cancers exhibited themethylated RASSFIA allele (FIG. 15; Table 7). By contrast, no methylatedalleles were detected in 104 paired resected nonmalignant lung tissues(FIG. 15; Table 7).

TABLE 7 Frequency of methylation-specific polymerase chain reactionassay for detection of RASSF1A CpU island- methylated alleles in lungand breast cancers No. No. of methylation alleles DNA sample source*tested (positive) (%) Primary resected NSCLCs 107 32 (30%) Correspondingnonmalignant lung 104 0 (0%) NSCLC lines 27 17 (63%) SCLC lines 47  47(100%) Primary resected breast cancers 39 19 (49%) Breast cancer lines22 14 (64%) *NSCLC = non-small-cell lung carcinoma; SCLC = small-celllung carcinoma.

The inventors found a high frequency of methylated 123F2 (RASSF1A)alleles in the panel of lung and breast cell cancer lines (Table 7).Because the lung and breast cancer cell lines represent essentiallyclonal populations of cancer cells without contaminating normal cells.the inventors tabulated the frequency of the methylated and unmethylated123F2 (RASSF1A) alleles (Table 8). While the lung and breast cancerlines often derive from clinically more aggressive lesions than theaverage population of tumors³⁴⁹⁻³⁵¹, the inventors previousstudies^(350, 351) have shown that cancer cell lines continue to retainthe genetic alterations found in the uncultured cancer specimens fromwhich they were derived. The presence of only the methylated allele isconsistent with either the methylation of both parental alleles or theretention of the methylated allele and the loss of the unmethylated 3pallele. All of the SCLC cell lines howed only the methylated allele orlacked 123F2 (RASSF1A) entirely because of a homozygous deletion,consistent with the nearly universal 3p21.3 allele loss inSCLC^(341, 350, 356). Of the NSCLC cell lines, 13 (48%) of 27 (Table 8)had only the methylated 123F2 (RASSF1A) allele, and 10 (37%) of 27 hadonly the unmethylated allele, consistent with a lower rate of 3p21.3allele loss in this tumor type³⁴¹. Likewise, 10 (45%) of 22 samples(Table 8) of breast cancer cell lines had only the methylated allele,and seven (32%) of 22 had only the unmethylated allele, again consistentwith the rate of 3p21.3 allele loss found in breast cancer³⁵¹. Asexpected, two tumor lines shown previously to have homozygous deletionsinvolving the 3p21.3 region were negative for both the methylated andthe unmethylated allele (Table 8)^(346, 347).

TABLE 8 Presence of methylated and unmethylated RASSF1A alleles in 97lung and breast cancer cell lines* RASSF1A CpG genotype MethylatedUnmethylated allele allele SCLC NSCLC BCCL Total + + 0 4 4  8 + − 47 1310 70 − + 0 10 7 17 − − 1 0 1  2^(†) Total 48 27 22 97 *SCLC =small-cell lung cancer~NSCLC = non-small-cell lung cancer; BCCL = breastcancer cell lines. ^(†)The two tumor cell lines withmethylation-specific polymerase chain reaction genotypes lacking bothmethylated and unmethylated alleles (SCLC line NCIH740 and breast cancerline 11CC 1500) were known to have homozygous deletions including theRASSFI locus in chromosome region 3p21.3.

For a subset of 61 lung and breast cancer cell lines, the inventorsperformed both expression and methylation analysis and found astatistically significant association (P<0.001, Fisher's exact test)between the presence of methylated RASSF1A alleles and the loss of 123F2(RASSF1A) expression. In 12 samples, 123F2 (RASSF1A) was expressed inthe absence of a methylated allele; in 44 samples, 123F2 (RASSF1A) wasnot expressed in the presence of a methylated allele; in four samples,123F2 (RASSF1A) was not expressed in the absence of methylated allele;and in one sample (a breast cancer cell line), 123F2 (RASSF1A) wasexpressed in the presence of both a methylated and an unmethylatedallele. These data show the critical association of 123F2 (RASSF1A)methylation with loss of 123F2 (RASSF1A) expression.

The inventors next assessed whether there was any association between123F2 (RASSF1A) promoter methylation and clinical findings in thepatients with primary NSCLC. The inventors found no statisticallysignificant association between 123F2 (RASSF1A) methylation and age,sex, tumor-node-metastasis (TNM) pathologic stage, or tumor histology in107 resected NSCLCs. In addition, the inventors found no statisticallysignificant association between 123F2 (RASSF1A) methylation and age, TNMpathologic stage, tumor histology, estrogen or progesterone receptorstatus, or HER2/Neu expression in 39 primary resected breast cancers.

Survival among lung cancer patients differed by the methylation statusof 123F2 (RASSF1A) (P=0.046) (FIG. 16). Also, by univariate analysis, inthis group of 107 patients with NSCLC treated with an attempt atcurative surgical resection, tumor (T1, T2, and T3), lymph node stage(N1 and N2), and reported weight loss were statistically significantpredictors of adverse survival. Neither smoking history (yes/no orpack-years with 40 pack-year cutoff) nor treatment differences (allpatients had surgical resection of lobectomy or pneumonectomy, and onlyfive had prior radiotherapy or chemotherapy) accounted for the adversesurvival. Because a multivariate analysis is of limited use with a smallsample size, the inventors performed a Cox proportional hazardsregression analysis by use of 123F2 (RASSF1A) methylation and the mainunivariate factors (tumor, lymph node stage, and weight loss). 123F2(RASSF1A) methylation was not found to be an independent prognosticfactor of survival. However, this result could be due to small numbersbecause even lymph node stage (a known prognostic factor) was also nolonger an independent factor in the analysis.

V. Effect of Exogenous Expression of 123F2 (RASSF1A) on Tumor CellPhenotype

The inventors examined the effect of RASSF1A on the tumor cell phenotypeby three methods. The inventors used anchorage-dependent colonyformation as a measure of proliferation and anchorage-independent colonyformation as a measure of malignant potential. The inventors alsodirectly assessed in vivo tumor formation.

The in vitro growth characteristics of NSCLC NCI-H1299 clones thatexpress RASSF1A were tested for anchorage-dependent andanchorage-independent (soft agar) growth. After 48 hours of growth innonselective medium, transiently transfected NSCLC NCI-H1299 cells weredetached with trypsin and diluted, usually 10- to 25-fold, incomplete-medium containing 800 μg/mL of G418 and plated into fresh100-mm dishes. The medium was changed twice weekly. After 14 days, themedium was removed, the plates were washed with phosphate-bufferedsaline (PBS), and the colonies were stained with 1% methylene blue in50% (vol/vol) ethanol. For the anchorage-independent, soft agar-growthassays, 1000 RASSFIA-expressing cells were suspended and plated in 0.33%Noble agar (Sigma Chemical Co. St. Louis, Mo.) in complete mediumsupplemented with 600 μg/mL G418 and layered over a 0.50% agar base incomplete medium. After 21 days, colonies greater than 0.2 mm in diameterwere counted.

For retrovirally infected cells, anchorage-independent growth assayswere performed as follows: 10000 viable selected cells from eachinfection were plated in 0.33% soft agar over a 0.50% agar base inDulbecco's modified Eagle medium (Life Technologies, Inc.) with 10%heat-inactivated fetal bovine serum. After 21 days, colonies greaterthan 0.2 mm in diameter were counted.

The inventors also tested the ability of RASSF1A-infected cells to growin vivo in nude mice. Male BALB/c nude (nu/nu) 3- to 6-week-old micewere irradiated on day 0 of the experiment in groups of five animals bya 5-minute exposure to 350 cGy from a cesium source. The next day, eachmouse was given an injection subcutaneously on its flank with 0.2 mL ofsterile PBS containing 10⁷ viable parental, vector control, or RASSF1Aretroviral-infected NSCLC NCI-H1299 tumor cells. Mice were monitoredevery 2-3 days for tumor size; once tumors reached greater than 1500mm³, the mice were killed.

The inventors first cloned RASSF1A cDNA into pcDNA3.1+, an expressionvector that contains a selectable marker, and transfected NCI-H1299cells, which lack endogenous 123F2 (RASSF1A) expression. After selectionfor 14-21 days, the inventors determined colony formation of NCI-H1299cells in both anchorage-dependent and anchorage-independent assays.Expression of 123F2 (RASSF1A) in NCI-H1299 cells resulted in a 40%-60%decrease in anchorage-dependent colony formation and in an approximate90% decrease in anchorage-independent colony formation compared withcells transfected with the pcDNA3. 1 vector alone (FIG. 17, A). BecauseNCI-H1299 cells have an intragenic p53 homozygous deletion, transientexpression of wild-type p53 can serve as a positive control for growthinhibition. Indeed, expression of wild-type p53 in NCI-H1299 cellsresulted in a 80% and 95% reduction in colony formation inanchorage-dependent and anchorage-independent assays, respectively (FIG.17, A). Several clones of NCI-H1299 cells transfected with 123F2(RASSF1A) were isolated in selective medium and were found to express123F2 (RASSF1A) by northern blot analysis (FIG. 17, B). Although theclones grew well in vitro, each had reduced anchorage-independent colonyformation by approximately 90% compared with the vector-transfectedcontrol clones (FIG. 17, C).

To eliminate the possibility that the pcDNA3.1+ vector mediated thegrowth-suppression effects, the inventors infected NCI-H1299 cells withretroviral-expression vectors containing 123F2 (RASSF1A) or RASSF1C andtested the ability of these cells to grow in an anchorage-independentmanner. Cells expressing 123F2 (RASSF1A) had a marked reduction in theability to form soft-agar colonies compared with cells infected with theretroviral empty vector or the retroviral vector containing RASSF1C(FIG. 17, D). Cells expressing the retroviral vector formed 3200colonies per 10000 cells plated. 123F2 (RASSF1A)-expressing cells formedonly 19% of the vector control colonies, while RASSF1C formed 108% ofthe vector control. RASSF1A- and RASSF1C-infected cells grew well invitro and showed no signs of toxicity or apoptosis.

Finally, the inventors tested the ability of the retrovirally infectedNCIH1299 cells to form tumors in nude mice. Cells transfected with thevector (parental cells) formed tumors rapidly (FIG. 17, E). By contrast,cells infected with 123F2 (RASSF1A) retroviral vector and expressing the123F2 (RASSF1A) protein had much lower tumorigenicity in vivo (FIG. 17,E).

Example 16 Several Genes in the Human Chromosome 3p21.3 HomozygousDeletion Region Exhibit Tumor Suppressor Activities In Vitro and In VivoI. Effects of Forced Expression of 3p Genes on Tumor Cell Growth.

To test the hypothesis that one or more of the 3p genes function astumor suppressors in vitro, the inventors performed a series ofexperiments to study the effects of expression of the 3p21.3 genes oncell proliferation in several types of Ad-3p-transduced human NSCLCcells and a normal HBEC line. Cells in each line were transduced invitro by Ad-101F6, Ad-FUS1, Ad-NPRL2, Ad-BLU, Ad-RASSF1, Ad-HYAL2 andAd-HYAL1 vectors at various MOIs in units of vp/c; cells were treatedwith PBS, Ad-EV, Ad-LacZ, or Ad-p53 as mock, negative, non-specific, orpositive controls, respectively. The transduction efficiency wasdetermined by examining GFP-expressing cells in the Ad-GFP-transducedcell population under a fluorescence microscope and was found to begreater than 80% at the highest MOI applied for each cell line.

Cell proliferation was analyzed by using the XTT assay to determine thenumber of viable cells remained at 1, 2, 3, and 5 days aftertransduction {only data for day 5 at highest MOIs (5000 vp/c for A549,1000 vp/c for H1299, 5000 vp/c for H460, 2500 vp/c for H358, and 1000vp/c for HBE, respectively) are shown} (FIG. 19). In all cases, theviability of transduced cells was compared with that of untransduced(PBS-treated) control cells (whose viability was set at 100%). As can beseen in FIG. 22, cell viability was significantly reduced in Ad-101F6-,Ad-Fus1-, and Ad-NPRL2-transduced A549 and H460 cells, which showhomozygousity for multiple 3p21.3 markers and contain wild-type p53, andH1299 cells, which exhibit 3p21.3 homozygous but also have a homozygousdeletion of p53. A modest reduction of cell viability was shown inAd-RASSF1C-transduced H1299 cells. However, no significant effect ongrowth was observed in any of these cells transduced with Ad-HYAL1,Ad-HYAL2, Ad-BLU, Ad-EV or Ad-LacZ. These results suggest that exogenousexpression of some wild-type 3p21.3 genes could inhibit 3p-deficienttumor cell growth or restore the tumor suppressor function of these3p21.3 genes in vitro.

To clarify the specificity of the observed inhibitory effects on tumorcell growth and examine the potential cytotoxicity of the exogenouslyexpressed 3p21.3 genes on normal cells, the inventors analyzed theeffects of these 3p21.3 genes on cell proliferation in 3p21.3heterozygous H358 cells and normal HBECs (FIG. 19). As shown in FIG. 19,HBECs transduced with all Ad-3p genes at highest MOIs had losses of cellviability of less than 10%, while H358 cells transduced with the samevectors had losses of cell viability less than 20% when compared withthe untransduced control cells. Similar levels of losses of cell numberswere observed in H358 and HBEC cells transduced with Ad-EV and Ad-LacZ.H358 cells which are deleted for p53 showed reduced cell viability whentransduced with the Ad-p53 control. These results couple with the lackof effect with Ad-LacZ, Ad-HYAL2, Ad-HYAL1, Ad-RASSF1, and Ad-BLU,demonstrate the specificity of the tumor-suppressing function of 3p21.3genes, FUS1, NPRL2, 101F6 in 3p-deficient tumor cells and indicate thatno generalized cytotoxicity was associated with exogenous expression ofthese wild-type 3p21.3 genes.

Expression of 3p21.3 genes in Ad-3p transfectants was verified byquantitative real-time RT-PCR, and known concentrations of human totalRNA and primers and TaqMan probe for β-actin DNA and for GAPDH cDNA wereused as standards and internal controls, respectively (FIG. 20). Thetranscription of FUS1 (FIG. 20A), 101F6 (FIG. 20B), NPRL2 (FIG. 20C),and HYAL1 (FIG. 20D) was demonstrated quantitatively by showing theassociation between increased levels of expression of these 3p21.3 geneswith increased MOIs of the corresponding Ad-3p vectors in transducedH1299 cells. The transcription of other 3p21.3 genes, HYAL2, HYAL1, BLU,and RASSF1, was also detected by real-time RT-PCR. The expression ofFUS1 and 101F6 proteins was detected also by western blot analysis usingavailable polyclonal antibodies raised against the oligopeptides derivedfrom their deduced amino acid sequences.

II. Induction of Apoptosis by 3p Genes in Ad-3p-Transduced Tumor Cells.

The ability of exogenously expressed 3p21.3 genes to induce apoptosis inthe Ad-3p-transduced H1299, A549, H460, H358, and HBEC cells wasanalyzed by FACS using the TUNEL reaction (FIG. 21). Induction ofapoptosis was detected in Ad-101F6-, Ad-FUS1-, and Ad-NPRL2-transducedA549 (FIG. 21A), H1299 (FIG. 21B), and H460 (FIG. 21C) cells, but not inH358 (FIG. 21D) and HBEC (FIG. 21E) cells. The apoptotic cellpopulations increased with increased duration of transduction; more than15-20%, 40-65%, and 75% of cells were apoptotic 5 days aftertransduction with Ad-101F6, Ad-FUS1, and Ad-NPRL2 in the transducedH1299, A549, and H460 cells, respectively, whereas only about 7% and 10%of cells treated with PBS alone and transduced with Ad-EV vector,respectively, were TUNEL-positive after the same time interval. Thelevels of apoptosis induction by Ad-101F6, Ad-FUS1, and Ad-NPRL2appeared 20-50% more significant in A549 and H460 cell lines withwild-type p53 genes (FIGS. 21A and 21C) than that in H1299 cell linedeleted for p53 gene (FIG. 21B). Levels of apoptosis in A549 and H460cells were comparable to those induced by Ad-p53 in p53-deficient H1299and H358 cells (FIGS. 21B and D). However, no significant induction ofapoptosis was observed in any tumor cell line transduced by Ad-BLU,Ad-RASSF1, Ad-HYAL2, and Ad-HYAL1 (FIG. 21). The levels and time ofinduction of apoptosis in cells transduced by these Ad-3p vectors werewell correlated with those of cell proliferation inhibition in cellstreated with the same vectors (FIG. 19), suggesting that suppression oftumor cell proliferation by 3p21.3 genes is mediated directly orindirectly through a mechanism of apoptosis induction.

III. Suppression of Tumor Growth by Intratumoral Injection of Ad-3pVectors.

To determine whether the observed inhibitory effects of these 3p21.3genes on tumor cell proliferation in vitro could be demonstrated ontumor growth in vivo, the inventors evaluated the efficacy of 3p21.3genes in suppressing tumor growth by direct intratumoral injection ofAd-3p21.3 gene vectors, along with PBS and Ad-EV, Ad-LacZ, and Ad-p53vectors as controls, into A549 or H1299 tumor xenografts in nu/nu mice(FIG. 22). The growth of tumors was recorded from the first injectionuntil 20 days after the last injection. Tumor volumes were normalized bycalculating the percentage increase in tumor volume after treatmentrelative to volume at the beginning of treatment in each group. In bothA549 (FIG. 22A) and H1299 (FIG. 22B) tumor models, all of the tumorstreated with Ad-101F6, Ad-FUS1, or Ad-NPRL2 showed significantlysuppressed growth (P<0.001), compared with mouse groups treated withAd-LacZ or Ad-EV controls, whereas no significant effect was observed inAd-BLU, Ad-RASSF1, and Ad-HYAL1-treated tumors. H1299 A549 tumorxenografts but not A549 H1299 tumors treated with Ad-HYAL2 showedsignificant reduction only at the end points of treatment (P=0.036).Moreover, a significantly stronger inhibition of tumor growth was shownin A549 tumors treated with Ad-101F6 and Ad-NPRL2 vectors than in tumorstreated with the Ad-p53 vector (FIG. 22A).

IV. Inhibition of Development of Experimental Lung Metastases byProtamine-Adenovirus Complex-Mediated 3p21.3 Gene Transfer.

A novel formulation using protamine/adenovirus complexes (designatedP-Ad) for enhanced systemic delivery of recombinant adenovirus in vivowas developed to further explore the potential of 3p21.3 genes insuppressing systemic metastases. An experimental A549 metastatic humanlung cancer model was used to study the effects of 3p21.3 gene transferon the development of lung metastases in nu/nu mice (FIG. 23). Theadenoviral 3p21.3 gene vectors were complexed to protamine and deliveredvia intravenous injection. The development of A549 metastases wassignificantly inhibited and the formation of metastatic tumor colonieson the surfaces of lungs from mice inoculated with A549 was reduced morethan 80% in animals treated with P-Ad-101F6, P-Ad-FUS1, P-Ad-NPRL2,P-Ad-BLUor P-Ad-HYAL2 compared with those in control treatment groups(FIG. 23A). However, no significant reduction of metastatic colonyformation was observed in animals treated with P-Ad-HYAL1 andP-Ad-RASSF1P-Ad-BLU. These data are consistent with results obtainedfrom Ad-3p-treated subcutaneous tumors, further supporting the roles ofthese 3p21.3 genes in suppression of tumor growth and inhibition oftumor progression in vivo.

Example 17 Overexpression of Candidate Tumor Suppressor Gene Fus1Isolated from the 3p 21.3 Homozygous Deletion Region Leads to G1 Arrestand Growth Inhibition of Lung Cancer Cells

Very frequent loss of one allele of chromosome arm 3p in both small lungcancer (SCLC) and non-small cell lung cancer (NSCLC) provides strongevidence for the existence of tumor suppressor genes (TSGs) in thischromosome region^(363; 36; 367; 371; 372). Multiple different 3pregions showing isolated allele loss were identified by detailedallelotyping studies suggesting there are several different TSGs locatedon 3p suggesting there are several different TSGs located on3p^(361; 362; 372). Nested homozygous deletions in lung cancer andbreast cancer cell lines have been found at 3p21.3 that focused oursearch on a 630 kb region including the identification, annotation, andevaluation of 25 new genes as TSGcandidates^(357; 365; 366; 368; 369; 370). A breast cancer deletionnarrowed this region further to 120 kb and 9 TSG candidates (CACNA2D2,PL6, 106F6, NPRL2/g21, BLU, RASSF1, FUS1, HYAL2, HYAL1) were located inor bordering this region³⁶⁹. One of these candidate TSGs, FUS1(AF055479), did not show homology with any known genes, was found tohave only few mutations in lung cancers, and usually was expressed atthe mRNA level in lung cancers³⁶⁶. Several NSCLCs (NCI-H322 andNCI-H1334) exhibited the same nonsense mutation, which arose fromaberrant mRNA splicing. This aberrant form lacked 28 bp of mRNA at the3′ terminus of FUS1 exon 2 resulting in a truncated predicted protein of82 amino acids compared to 110 amino acids in the wild-type (FIG. 24).To confirm the inventors mutational analysis, which previously had beenconducted on lung cancer cell line DNAs, they searched for othermutations in FUS1 in primary uncultured lung cancers. Single strandconformation polymorphism (SSCP) analysis was performed using genomicDNA of 40 primary uncultured lung cancers (9 SCLCs and 31 NSCLCs) (FIG.24)³⁶⁰. No mutations were detected although the inventors found a singlenucleotide polymorphism in intron 2 that did not alter the amino acidsequence of FUS1.

The inventors next considered CpG island promoter region methylation asan epigenetic mechanism leading to TSG inactivation. In fact, such tumoracquired promoter region methylation was found to occur for the RASSF1AmRNA isoform residing immediately centromeric to FUS1^(354; 358).However, FUS1A mRNA was expressed in most lung cancers making such CpGmethylation an unlikely method of inactivation of FUS1³⁶⁶. In addition,the 5′ putative promoter region containing CpG islands of FUS1 wassequenced using sodium bisulfite treated³⁵⁵ DNA from 6 lung cancers werethe inventors did not detect FUS1 protein expression and found no CpGmethylation.

The possibility of haploinsufficiency or reduced expression of FUS1 wasconsidered as another mechanism for this gene to participate in lungcancer pathogenesis^(356; 359; 373). The inventors first performedwestern blot analysis of a panel of lung cancer cell lines using ananti-Fus1 anti peptide antibody which readily detected exogenouslyexpressed Fus1 (FIG. 25) but could not detect any endogenous FUS1expression in lung cancers (FIG. 25 for H1299 NSCLC given as an exampleof negative data). This lack of detection could be due to a variety offactors including the quality of the antibodies. Nevertheless, if lossor low levels of FUS1 protein expression was involved in lung cancerpathogenesis the inventors reasoned that exogenous introduction andexpression of Fus1 might suppress the malignant phenotype. Colonyformation assays were performed after transfection of FUS1 expressionvectors. The inventors made a C terminal FLAG-tagged FUS1 construct byPCR and ligated it into expression vector pcDNA3.1 (Invitrogen, CarlsbadCalif.). Empty vector and an expression vector containing wild-typeFUS1, FLAG-tagged FUS1, and the 82 aa mutant FUS1 werewas transfectedinto NSCLC NCI-H129.9 cells which has suffered allele loss for the3p21.3 630 kb region and does not express detectable FUS1 protein (FIG.25), and NSCLC NCI-H322 cells containing a expressing theendogenoushomozygous nonsense truncation mutation of FUS1 1 and also notexpressing detectable FUS1 protein. Expression of the FUS1 constructs inH1299 cells after transient transfection was confirmed by Western blotanalysis using anti-Flag and anti-N terminal FUS1 antibodies (FIG. 25).The effect of FUS1 transfection with a neo resistance gene on lungcancer colony formation was tested. The numbers of G418 resistantcolonies in the FUS1 transfections were dramatically reduced incomparison with transfection with the empty vector (FIG. 25). Bycontrast, the number of colonies formed in the mutant FUS1 transfectantswas only slightly reduced, suggesting that this lung cancer-associatedmutant FUS1 was functionally inactive (FIG. 25).

An ecdysone inducible mammalian expression system in H1299 cells wasdeveloped to confirm that overexpression of FUS1 could inhibit tumorcell growth. In this system, FUS1 expression is induced in the presenceof Ponasterone A. H1299 parent ECR9 cells with the regulatable hormonereceptor vector pVgRXR alone served as an additional control. H1299 ECR9cells were transfected with pINDsp1-FUS1-FLAG(neo), selected with G418in the presence or absence of Ponasterone A, and compared the numbers ofG418 resistant colonies. The number of colonies formed in cells withinduced expression of in the FUS1 induced condition was decreased anaverage of 75±8% compared with number of colonies in cells under theuninduced condition, providing another confirming ation of the growthinhibitory activity of FUS1. Twenty stable G418 resistant clones wereisolated in the uninduced condition and, the inducible expression ofFUS1-FLAG was examined. Among them, 6 clones showed some FUS1 inductionand two stable clones were selected (Cl.13 and Cl.16) in whichexpression of FUS1-Flag was as clearly inducible by Ponasterone A (FIG.26). However, both cell lines expressed some FUS1 in the uninducedcondition, indicating that regulation of FUS1 expression was leaky.

The cell growth rate was examined in induced and uninduced conditions bythe MTT assay. Ponasterone A has no effect on the growth of parentalcell line H1299 ECR 9 cells, but the growth of Cl. 13 and Cl. 16 cellswere inhibited in the presence of Ponasterone A (FIG. 26). The inductionof Fus1 expression and inhibition of tumor cell growth appeared to bedependent on the dose of Ponasterone A both increasing with theincreased concentrations of Ponasterone A (FIG. 26). With Fus1induction, the doubling times of the tumor cells were also increased inboth clones, from 22 to 46 hrs for Cl. 13 and from 21 to 45 hrs forC1.16, respectively. These results also indicated that overexpression ofFus1 suppresses H1299 lung cancer cell growth in vitro.

An increase of apoptosis in H1299 cells under induced condition by TUNELassay was not observed. However, when cells were induced by PonasteroneA to express Fus1 for 48 hrs and analyzed by fluorscent activated cellsorter (FACS) analysis (see legend of FIG. 26 for details) by FACSanalysis the inventors found: parental H1299-ECR9 cells to haveunchanged cell cycle parameters (G1 51%, S 18%, G2/M 31% uninduced andG1 50%, S 18%, G2/M 32% induced); while Fus1 induced clones showed G1arrest (H1299 clone13 showed G1 50%, S 17%, G2/M 33% uninduced and G165%, S 10%, G2/M 25% induced; and H1299 clone16 G1 56%, S 16%, G2/M 28%uninduced and G1 65%, S 12%, G2/M 23% induced). The increase in G1% wassignificant (P<0.05, students t test). These results suggest of cellcycle analysis showed that overexpression of FUS1 in H1299 cells isassociated with G1 arrest and alteration of cell cycle kinetics.

Lung cancer cell lines do not express detectable endogenous levels ofFus1 protein, and exogenous introduction of Fus1 with overexpressioninhibited lung cancer cell growth in vitro. This growth inhibition wasseen in a lung cancer line suffering allele loss for the region and inanother carrying a homozygous truncating mutation of FUS1. In addition,the inventors found that this truncated Fus1 protein had lost tumorgrowth suppressing activity. Besides the acute transfection studies, theinventors established a Fus1 inducible system and showed that tumorgrowth inhibition was correlated with the level of expression of Fus1protein. In addition, cell cycle analysis using the sameexpression-regulatable system showed that the mechanism for theinhibition of cell growth was associated with G1 arrest and not withinduction of apoptosis. Finally, the inventors confirmed that somaticmutation of FUS1 was rare in primary lung cancers (0/40), in agreementwith previous studies which showed 3/79 lung cancers with alterations inthe FUS1 gene (2 nonsense mutations and 1 deletion). In fact thefrequency of mutation in any of the 22 out of 25 candidate genes theinventors have studied in detail in this 600 kb 3p21.3 region is lowcompared to the high frequency LOH at this locus. One possibility toaccount for the low mutation frequency is loss of expression of FUS1 orother of the 3p21.3 genes by tumor promoter acquired methylation. Theexpression of RASSF1A mRNA isoform isolated from the same 3p21.3deletion region and 15.5 kb centromeric of FUS1 was repressed in manylung cancers by acquired CpG island promoter DNA methylation for thisgene^(354; 358). Replacement of RASSF1A inhibited tumor cell growth invitro and in vivo indicating RASSF1A is another candidate tumorsuppresser gene in this locus. However the inventors have not found lossof FUS1 mRNA expression³⁶⁶ or 5′ region CpG methylation for FUS1 in lungcancers thus excluding tumor acquired promoter methylation as aninactivating mechanism for the FUS1 gene. FUS1 may act ashaploinsufficient tumor suppressor gene³⁵⁶. The inventors experimentsshowed that overexpression of FUS1 caused G1 arrest in H1299. Althoughsome signal or environmental cue may induce the expression of Fus1 andlead to G1 arrest in normal cells, 3p allelic loss and some otheralteration of FUS1 in malignant cells may lead to haploinsufficiencyand/or loss of expression of FUS1 in lung tumors and escape from cellcycle arrest.

All of the compositions and/or methods disclosed and claimed herein canbe made and executed without undue experimentation in light of thepresent disclosure. While the compositions and methods of this inventionhave been described in terms of preferred embodiments, it will beapparent to those of skill in the art that variations may be applied tothe compositions and/or methods and in the steps or in the sequence ofsteps of the method described herein without departing from the concept,spirit and scope of the invention. More specifically, it will beapparent that certain agents which are both chemically andphysiologically related may be substituted for the agents describedherein while the same or similar results would be achieved. All suchsimilar substitutes and modifications apparent to those skilled in theart are deemed to be within the spirit, scope and concept of theinvention as defined by the appended claims.

REFERENCES

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference:

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1.-62. (canceled)
 63. A method for suppressing growth of a tumor cellcomprising contacting said cell with an expression cassette comprising:(a) an isolated polynucleotide encoding a human Gene 21 (NPRL2)polypeptide; and (b) a heterologous promoter active in said tumor cell,under conditions permitting the uptake of said polynucleotide by saidtumor cell.
 64. The method of claim 63, wherein said tumor cell isderived from a tumor selected from the group consisting of brain tumor,lung tumor, liver tumor, spleen tumor, kidney tumor, lymph node tumor,small intestine tumor, blood cell tumor, pancreatic tumor, colon tumor,stomach tumor, cervix tumor, breast tumor, endometrial tumor, prostatetumor, testicle tumor, ovarian tumor, skin tumor, head and neck tumor,esophageal tumor, oral tissue tumor, and bone marrow tumor.
 65. Themethod of claim 63, wherein said polynucleotide is contained in a viralvector.
 66. The method of claim 65, wherein said viral vector is aretroviral vector, an adenoviral vector, an adeno-associated viralvector, a vaccinia viral vector, or a herpesviral vector.
 67. The methodof claim 66, wherein said viral vector is an adenoviral vector.
 68. Themethod of claim 63, wherein said polynucleotide is contained in aliposome.
 69. A method of altering the phenotype of a tumor cellcomprising contacting said cell with an expression cassette comprising:(a) an isolated polynucleotide encoding a human Gene 21 (NPRL2)polypeptide; and (b) a heterologous promoter active in said tumor cell,under conditions permitting the uptake of said polynucleotide acid bysaid tumor cell.
 70. The method of claim 69, wherein the phenotype isselected from the group consisting of proliferation, migration, contactinhibition, soft agar growth, cell cycling, invasiveness, tumorigenesis,and metastatic potential.
 71. The method of claim 69, wherein saidpromoter is a cytomegalovirus (CMV) promoter.
 72. A method of treatingor inhibiting a cancer in a subject suffering therefrom comprisingadministering to said subject an expression cassette comprising: (a) anisolated polynucleotide encoding a human Gene 21 (NPRL2) polypeptide;and (b) a heterologous promoter active in tumor cells of said subject,whereby expression of said Gene 21 (NPRL2) polypeptide inhibits saidcancer.
 73. The method of claim 72, wherein said subject is a human.74.-83. (canceled)
 84. The method of claim 72, wherein said cancer isselected from the group consisting of brain cancer, lung cancer, livercancer, spleen cancer, kidney cancer, lymph node cancer, small intestinecancer, blood cell cancer, pancreatic cancer, colon cancer, stomachcancer, cervix cancer, breast cancer, endometrial cancer, prostatecancer, testicle cancer, ovarian cancer, skin cancer, head and neckcancer, esophageal cancer, oral tissue cancer, and bone marrow cancer.85. The method of claim 72, wherein said expression cassette iscontained in a viral vector.
 86. The method of claim 85, wherein saidviral vector is a retroviral vector, an adenoviral vector, anadeno-associated viral vector, a vaccinia viral vector, or a herpesviralvector.
 87. The method of claim 86, wherein said viral vector is anadenoviral vector.
 88. The method of claim 72, wherein said expressioncassette is contained in a liposome.
 89. The method of claim 72, whereinsaid expression cassette further comprises a poly-A sequence.
 90. Themethod of claim 89, wherein said poly-A sequence is bovine growthhormone (BGH) poly-A sequence.
 91. The method of claim 72, wherein saidexpression cassette is administered intratumorally, in the tumorvasculature, local to the tumor, regional to the tumor, or systemically.92. The method of claim 72, further comprising administering achemotherapuetic agent to said subject.
 93. The method of claim 92,wherein said chemotherapeutic comprises cisplatin (CDDP), carboplatin,procarbazine, mechlorethamine, cyclophosphamide, camptothecin,ifosfamide, melphalan, chlorambucil, busulfan, nitrosurea, dactinomycin,daunorubicin, doxorubicin, bleomycin, plicomycin, mitomycin, etoposide(VP16), tamoxifen, raloxifene, estrogen receptor binding agents, taxol,gemcitabien, navelbine, farnesyl-protein tansferase inhibitors,transplatinum, 5-fluorouracil, vincristin, vinblastin and methotrexate.94. The method of claim 72, further comprising administering radiationto said subject.
 95. The method of claim 94, wherein said radiation isdelivered local to a cancer site.
 96. The method of claim 94, whereinsaid radiation is whole body radiation.
 97. The method of claim 94,wherein said radiation comprises γ-rays, X-rays, accelerated protons,microwave radiation, UV radiation or the directed delivery ofradioisotopes to tumor cells.
 98. The method of claim 72, furthercomprising administering a second anticancer gene to said subject. 99.The method of claim 98, wherein said second anticancer gene is a tumorsuppressor.
 100. The method of claim 98, wherein said second anticancergene is an inhibitor of apoptosis.
 101. The method of claim 98, whereinsaid second anticancer gene is an oncogene antisense construct.102.-117. (canceled)
 118. The method of claim 72, wherein saidheterologous promoter is a cytomegalovirus (CMV) promoter.
 119. Themethod of claim 72, wherein said tumor cells lack expression of afunctional Gene 21 (NPRL2) polypeptide.
 120. The method of claim 72,wherein said tumor cells comprise hypermethylation of Gene 21 (NPRL2)gene promoter.
 121. The method of claim 63, wherein said expressioncassette is contained in a lipid preparation.
 122. The method of claim69, wherein said expression cassette is contained in a lipidpreparation.
 123. The method of claim 72, wherein said expressioncassette is contained in a lipid preparation.
 124. The method of claim121, wherein said liposome is comprised ofN-(1-[2,3-Dioleoyloxy]propyl)-N,N,N-trimethylammonium (DOTAP) andcholesterol.
 125. The method of claim 122, wherein said liposome iscomprised of N-(1-[2,3-Dioleoyloxy]propyl)-N,N,N-trimethylammonium(DOTAP) and cholesterol.
 126. The method of claim 123, wherein saidliposome is comprised ofN-(1-[2,3-Dioleoyloxy]propyl)-N,N,N-trimethylammonium (DOTAP) andcholesterol.